0432
As bait the sequences encoding the MEINOXSTM domain (amino acids 116–220 of the STM protein) or the slightly longer MEINOX-ELKSTM (amino acids 116–287) domain were cloned into the vector pGBKT7 (Clontech) and expressed in fusion with the GAL4 DNA-binding domain.
0809
Verified cDNA inserts were either directly transferred into appropriate secondary vectors for bimolecular fluorescence complementation (BiFC), co-immunoprecipitation or transgenic experiments and served as templates in second-round PCR-amplification to remove the stop codon or to adjust open reading frames (ORFs) for translational fusions.

0019
Verified cDNA inserts were either directly transferred into appropriate secondary vectors for bimolecular fluorescence complementation (BiFC), co-immunoprecipitation or transgenic experiments and served as templates in second-round PCR-amplification to remove the stop codon or to adjust open reading frames (ORFs) for translational fusions.
0019
HA-tagged (1/3 aliquot) partner proteins were identified using an anti-HA horseradish peroxidase (HRP)-conjugated antibody (clone 3F10; Roche 2012819).
0019
In addition to the interactions in yeast, we performed co-immunoprecipitation experiments with epitope-tagged full-length proteins. The HA-tagged BLH proteins shown in Figure 2B were used to co-precipitate epitope-tagged STM-His (Figure 2C).

0019
The co-immunoprecipitation experiments therefore confirm that the full-length STM protein interacts with full-length ATH1, BLH3 and BLH9 proteins in vitro and substantiate the affinity of BLH/STM interactions.
0809
According to our BiFC results, however, STM is targeted into the nucleus as a heterodimer with ATH1, BLH3 and BLH9.
0416
(F–H) Z-stack projection of a median series through the nucleus performed with confocal laser scanning microscope in leak epidermal cells.
0019
STM/BLH interactions in yeast and co-immunoprecipitations.

0432
(A) Galactosidase activity mediated by the interaction of the MEINOX domain cloned into pGBKT7 (bait) with BLH partner proteins expressed in pACT2.

0019
Asterisks mark control lanes showing that STM-His is undetectable in immunoprecipitations in the absence of BLH partner proteins.
0809
(A–C) BiFC staining of the nucleus obtained after coexpression of STM/BLH constructs as indicated above each photograph in leek epidermal cells.

0809
(G) Schematic representation of BLH3 and STM deletion constructs fused to the YFP NYFP- or CYFP- sub-domains.
0114
A 5-Å gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the CH2-CH3 interface, the binding site for several mammalian and bacterial Fc-binding proteins.
0402
The Fc proteins were immobilized on the surface of a biosensor chip, and the binding of CgE was assayed at pH 8 and pH 6.

0019
The relatively low affinity of the CgE/Fc interaction likely explains why a CgE/Fc binding interaction was not detectable by coimmunoprecipitation analyses [16].
0114
The complex crystals could not be improved by changing the gE-gI or Fc construct or by manipulating the crystals (summarized inMaterials and Methods). A 5.0-Å native data set was collected from the best of several hundred crystals (Table S2) and used for molecular replacement with the IgG Fc (pdb entry 1dn2) and CgE structures as search models. A molecular replacement solution was obtained when the Fc was positioned first followed by two CgE molecules, one bound to each Fc chain.
0114
The molecular replacement solution positions a CgE molecule near the CH2-CH3 linker on each of the Fc chains to make an approximately two-fold symmetric 2:1 gE-gI complex (Figure 3A). The model allows sufficient space in the unit cell for gI and NgE, which are unaccounted for in the molecular replacement solution, and shows a plausible packing arrangement including contacts between crystallographically related CgE molecules. Other features of the molecular replacement solution suggest that it is correct.
0114
We used experimental phasing methods to independently corroborate the molecular replacement model. A screen of complex crystals that had been soaked in various heavy-atom compounds resulted in four approximately isomorphous data sets (Table S2). An initial analysis of a KIrCl3 derivative identified five iridium sites located near the Fc molecule as positioned in the molecular replacement solution, four of which are related by the Fc dimer symmetry and two of which make a chemically-plausible interaction with a methionine (Met358) on each half of the Fc dimer (Figure 3B).

0114
An overlay of the molecular replacement model with a 5-Å heavy atom–phased electron density map (Figure S2) shows overlap of the model with electron density, including density for theN-linked carbohydrates attached to the CH2 domain of Fc, and extra density that likely corresponds to gI and NgE. Thus, experimental phasing methods using a combination of heavy-atom derivatives and SeMet-substituted crystals provides further confirmation of the molecular replacement solution and validates its use as a model for the CgE/Fc interaction in the context of the gE-gI/Fc complex.
0114
Of the five resulting models for the CgE/Fc interaction, one is similar to the crystallographic model obtained by molecular replacement and experimental phasing methods (Figure 3C).
0114
Crystal structures are available for Fc complexed with four other proteins, domain B1 of protein A [25], domain C2 of protein G [28], rheumatoid factor [24], and FcRn [27], and with one peptide that bind to this region [19].
0114
Although we cannot rule out local conformational variability that occurs as a function of pH, it seems unlikely that CgE undergoes large changes in conformation due to its relative compactness as a single domain, and arguments against pH-induced conformational changes in Fc have been made based on the similarity of Fc crystal structures at pH values between 4.1 and 8.0 [27].
0114
Because we were able to solve the crystal structure of the CgE subunit of the gE-gI ectodomain at high resolution, the identification of CgE as a minimal Fc-binding domain (Figure 1) made its structure useful for interpreting the low-resolution gE-gI/Fc complex structure. An independent prediction of the complex structure using the coordinates of the unbound CgE and Fc molecules revealed a close agreement with the experimentally determined gE-gI/Fc structure (Figure 3C), demonstrating the power of new protein docking methods [33,34], and their potential for facilitating difficult crystallographic problems.
0114
As revealed in the low resolution crystal structure of a gE-gI/Fc complex, the CgE portion of gE-gI binds to Fc at the CH2-CH3 interdomain junction (Figure 3A), consistent with previous studies of the gE-gI/Fc interaction [12,21,23] and allowing a comparison of the Fc-binding properties of CgE with other proteins that bind to the same site on Fc [19,20,27].
0114
The model for CgE binding to Fc derived from the crystal structures of CgE and a gE-gI/Fc complex can be used to gain insight into the regions of CgE that are implicated in cell-to-cell spread of HSV.
0071
Bound protein was eluted in buffer containing 40 mM Tris (pH 8), 300 mM NaCl, and 250 mM imidazole, and further purified by size-exclusion chromatography on a Superdex 75 HiLoad 16/60 column (GE Healthcare, Piscataway, New Jersey, United States) that was equilibrated in 10 mM HEPES (pH 7.6) and 150 mM NaCl.
0402
In this system, binding between a molecule coupled to a biosensor chip (the “ligand”) and a second molecule injected over the chip (the “analyte”) results in changes in the surface plasmon resonance signal that are read out in real time as resonance units [54]. wtFc and nbFc, both derived from human IgG1, were purified from CHO cell supernatants as described previously [12] and immobilized on a CM5 biosensor chip (Biacore) using primary-amine coupling as described by the manufacturer.
0114
Microseeding increased the frequency of obtaining single crystals. SeMet-substituted CgE crystals were grown in conditions similar to the native crystals with the addition of 10 mM MgCl2 in the well solution, which decreased the disorder that was often observed in the diffraction of the native crystals. All crystals were cryoprotected in well solution with an additional 15% PEG 4000, added in 5% increments, and stored in liquid nitrogen prior to data collection at −180 °C.
0114
Complex crystals were prepared for data collection by transferring into drops containing either increasing concentrations of sodium malonate in 0.2 M increments up to 2 M, or 1.4 M sodium malonate plus increasing concentrations of glycerol in 5% (vol/vol) increments up to 15% and then stored in liquid nitrogen prior to data collection at −180 °C. Hundreds of crystals were screened for diffraction using synchrotron radiation at Stanford Synchrotron Radiation Laboratory beamlines 9–1 and 9–2. The best crystal diffracted anisotropically to ˜4.2 Å along thel axis and ˜5.4 Å along theh andk axes. Complex crystals were also screened for the effects of various cryoprotectants, crystallization additives, enzymatic deglycosylation ofN-linked andO-linked sugar moieties on gE-gI, dehydration conditions, and annealing protocols on the diffraction; however, none of these variables significantly improved the quality of the diffraction.
0114
For heavy-atom screening, complex crystals were soaked in 38 different heavy-atom compounds for between 18 h to 7 d, back-soaked for 2–3 h during cryopreservation, and screened for diffraction and fluorescence at the appropriate energies. Four datasets, which were collected near thef′′ peak energies for crystals that were soaked in KIrCl3, PIP, EMP, or Na2WO4, were isomorphous with the native data set (Table S2).
0114
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure. X-ray crystallography is better suited to elucidate the structures of large proteins and accurately define interfaces of protein complexes, but requires crystallization, which can yield artifacts due to crystal packing.

0077
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure.

0077
NMR spectroscopy can be performed in solution, and low-resolution information on the conformational and aggregation states of proteins can be quickly obtained even for large species using heteronuclear NMR experiments such as1H-15N heteronuclear single quantum coherence (HSQC).

0114
In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.

0077
In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.
0114
Correspondingly, initial screens yielded no crystals for Munc13–13–150 and Munc13–13–209, and extensive screens with Munc13–13–132 only led to needle-like crystal clusters that were not suitable for structure determination (Figure S1).

0114
This fragment readily yielded crystals under 20 different conditions of a basic crystallization screen (Index screen, Hampton Research, Aliso Viejo, California, United States). After condition optimization, we obtained high-quality crystals (Figure S1) that allowed us to solve the crystal structure of the Munc13–1 C2A-domain homodimer at 1.44 Å resolution using molecular replacement.Table 1 describes the structural statistics andFigure 2A shows a representative portion of the electron density.
0114
However, this β-hairpin is well packed against strand 6 of monomer C itself and appears to be an intrinsic feature of the Munc13–1 C2A domain, because the two Munc13–1 monomers present in the crystal structure of the Munc13–1/RIM2 heterodimer also contain this β-hairpin (see below).
0114
Hence, we designed two charge-reversal mutations, K32E and E63K, to disrupt Munc13–1 homodimerization based on the crystal structure of the C2A-domain homodimer.
0114
All these results strongly suggest that the monomer A/RIM2α82–142 complex observed in the crystals faithfully reflects the true Munc13–1/RIM2α binding mode, whereas the presence of a second Munc13–13–150(K32E) molecule in the crystals must be considered a consequence of crystal packing.
0114
Our Munc13–13–150(K32E)/RIM2α82–142 structure, together with the crystal structure of a rabphilin/Rab3A complex [27], and the sequence homology between rabphilin and RIM2α, also provide an explanation for our previous observations that Munc13–1, RIM2α, and Rab3A form a tripartite complex and that Munc13–1 alters the RIM2α/Rab3A binding mode [20].
0114
In the past, NMR spectroscopy and X-ray crystallography were largely viewed as alternative methods for structure determination of biomolecules, but the usefulness of combining the strengths of both techniques is increasingly being recognized [32,33].

0077
In the past, NMR spectroscopy and X-ray crystallography were largely viewed as alternative methods for structure determination of biomolecules, but the usefulness of combining the strengths of both techniques is increasingly being recognized [32,33]. On the other hand, two structural genomics studies recently indicated that there is no overt correlation between1H-15N HSQC spectral quality and successful crystallization of protein targets [34,35]. However, it is unclear to what extent the fragment length of each target was optimized in these studies, and a separate structural genomics effort suggested that NMR spectra can be used to identify promising targets for structure determination by X-ray crystallography [36]. The data presented here, together with our previous NMR analysis of Munc13–1/α-RIM/Rab3 interactions [20], provide a particularly compelling illustration of how NMR spectroscopy can assist in X-ray diffraction studies of protein complexes that present particularly challenging problems for crystallization, and at the same time can provide complementary information. Thus,1H-15N HSQC spectra were instrumental to map the regions involved in these interactions, to identify sequences of the complexes that can hinder crystallization because they are unstructured and promote aggregation, to interpret the X-ray results, and to resolve ambiguities. Altogether, these observations suggest that combining the strengths of NMR spectroscopy in fragment optimization and analysis of protein–protein interactions in solution with the high accuracy of structure determination by X-ray crystallography for biomolecules of any size will be particularly useful to study complex protein networks.

0114
However, it is unclear to what extent the fragment length of each target was optimized in these studies, and a separate structural genomics effort suggested that NMR spectra can be used to identify promising targets for structure determination by X-ray crystallography [36]. The data presented here, together with our previous NMR analysis of Munc13–1/α-RIM/Rab3 interactions [20], provide a particularly compelling illustration of how NMR spectroscopy can assist in X-ray diffraction studies of protein complexes that present particularly challenging problems for crystallization, and at the same time can provide complementary information.

0114
Altogether, these observations suggest that combining the strengths of NMR spectroscopy in fragment optimization and analysis of protein–protein interactions in solution with the high accuracy of structure determination by X-ray crystallography for biomolecules of any size will be particularly useful to study complex protein networks.
0096
Unlabeled and isotopically labeled proteins were expressed in bacteria as GST fusions as described [20]. The fusion proteins were isolated on glutathione-Sepharose beads (Amersham Biosciences, Little Chalfont, United Kingdom), cleaved from the GST moiety, and further purified by size-exclusion or ion-exchange chromatography.

0226
The fusion proteins were isolated on glutathione-Sepharose beads (Amersham Biosciences, Little Chalfont, United Kingdom), cleaved from the GST moiety, and further purified by size-exclusion or ion-exchange chromatography.

0071
Gel-filtration binding experiments were performed on Superdex S75 or S200 columns (Amersham) in 30 mM Tris-HCl buffer containing 150 mM NaCl, and 1 mM Tris(2-carboxyethyl)-phosphine (TCEP) at pH 7.4.
0065
ITC experiments were performed using a VP-ITC system (MicroCal, Northampton, Massachusetts, United States) at 20 °C in a buffer composed of 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP.

0065
Data were fit with a non-linear least-squares routine using a single-site binding model with Origin for ITC v.5.0 (Microcal), varying the stoichiometry(n), the enthalpy of the reaction (ΔH) and the association constant (Ka).
0077
1H-15N HSQC spectra were acquired at 25 °C on Varian INOVA500, INOVA600, or INOVA800 spectrometers (Varian, Palo Alto, California, United States) using H2O/D2O 95:5 (v/v) as the solvent.
0114
Prior to data collection, crystals were first transferred into 0.02 M sodium acetate buffer (pH 5.5), then transferred step by step to a series of solutions containing 0.02 M sodium acetate (pH 5.5) with increasing concentration of ethylene glycol (5%, 10%, 15%, 20%, 25%, and 30% [v/v] respectively), and finally flash-cooled in liquid propane. Diffraction data were collected at the Structural Biology Center beamline 19ID of the Advanced Photon Source at 100 K to a Bragg spacing (dmin) of 1.44 Å. The crystals exhibited the symmetry of space groupP21, contained four molecules per asymmetric unit, and had unit cell parameters ofa = 43.56 Å,b = 127.14 Å,c = 50.74 Å, and β = 90.27°.

0114
Prior to data collection, crystals were transferred into a solution of 1.4 M ammonium tartrate (pH 7.0) and 20% (v/v) ethylene glycol, and then flash-cooled in liquid propane. Diffraction data were collected at the Structural Biology Center beamline 19ID of the Advanced Photon Source at 100 K to a Bragg spacing (dmin) of 1.78 Å. The crystals exhibited the symmetry of space groupP212121, and had unit cell parameters ofa = 50.25 Å,b = 93.53 Å,c = 113.13 Å.
0071
(B) Gel filtration analysis of Munc13–13–150 (black), RIM2α82–142 (blue), and the complex between them (red).
0077
(C)1H-15N HSQC spectrum of15N-labeled Munc13–13–150 at 500 MHz.
0077
(D)1H-15N HSQC spectrum of15N-labeled Munc13–13–150 bound to unlabeled RIM2α82–142 at 500 MHz.
0071
(A) Gel filtration analysis of Munc13–13–150(K32E) (black), RIM2α82–142 (blue), and the complex between them (red).
0065
(B) ITC analysis of the binding of Munc13–13–150(K32E) to RIM2α82–142.
0077
(C)1H-15N HSQC spectra of15N-labeled Munc13–13–150(K32E) alone (black) and bound to unlabeled RIM2α82–142 (red) at 800 MHz.
0077
(D)1H-15N HSQC spectra of the15N-Munc13–13–150(K32E)/RIM2α82–142 complex (red) and the wild-type15N-Munc13–13–150/RIM2α82–142 complex (black) at 800 MHz.
0019
1 B) and endogenous integrins that were immunoprecipitated with antibodies against α1, α2, α5, α6, and β1 subunits (Fig.

0019
These associations were specific, as Rab7 and Rab11 failed to coprecipitate β1-integrin from these cells, even though separately all proteins were efficiently immunoprecipitated (Fig.
0432
(D) α2- and α11-integrin cytoplasmic domains and their point mutants (indicated in bold) were cloned as Gal4BD fusions and used as baits with Rab21 (amino acids 95–222) Gal4AD prey in yeast two-hybrid assays.
0019
Immunoprecipitations (IPs) were performed as indicated, and the Rab coprecipitating proteins were detected first with anti-biotin antibody followed by stripping and reprobing with anti–β1-integrin antibody.
0027
In the lighter fractions (3–5), GFP-Rab21 and integrin were found to cosediment with the Golgi-marker GM130, whereas in the denser fractions, cosedimentation was observed with the ER marker P115 (fractions 6–8) and EEA1 (fractions 7–9; Fig.
0040
Further characterization of the large intracellular structures induced by GFP-Rab21 overexpression was performed by electron microscopy and immunogold labeling of GFP (Fig.
0416
Combined total internal reflection fluorescence microscopy (TIRFM; pseudocolored green) and conventional widefield epifluorescence analysis (pseudocolored red) showed GFP-Rab21 vesicles emanating from the membrane into the cell (changing from green to red/yellow) and back or vice versa (although formally this technique does not exclude the possibility that some vesicles will by chance come closer and further away from the plasma membrane).
0416
(B and C) Combined widefield epifluorescence (pseudocolored red) and TIRFM (pseudocolored green) analysis of GFP-Rab21WT (B; see Video 2) or GFP-Rab21GDP (C; see Video 3) transfected cells.
0416
(B and C) MDA-MB-231 cells transfected with GFP–α2-integrin and Rluc-Rab21 (B) or with GFP–α2-integrin alone (C) were plated on collagen for 1 h, and time-lapse series were acquired with widefield epifluorescence microscopy. Cotransfected cells (B; see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200509019/DC1) show green fluorescent vesicles moving in the cytosol.
0054
(C) Cell surface expression of β1-integrin was analyzed with dual-color FACS from transiently transfected MDA-MB-231 cells.

0416
(F) Subcellular localization of GFP-integrin in the transfected collagen adhering cells was studied by widefield fluorescence microscopy.
0077
Evidence from detailed biochemical and nuclear magnetic resonance studies (Stefansson et al., 2004; Vinogradova et al., 2004) indicates that upon integrin activation the membrane-proximal regions of the integrin cytoplasmic tails move out of the membrane into the cytoplasm, revealing the highly conserved residues to the cytoplasmic face and involving substantial structural changes in the cytoplasmic tails.
0019
For immunoprecipitations with endogenous proteins, confluent MDA-MB-231 cells (20 × 106) were collected from plastic plates with cold PBS.

0019
Postcentrifugation supernatant was precleared with BSA-blocked (IP buffer) protein G–agarose beads and divided into five aliquots for immunoprecipitations with different anti-Rab antibodies or a control antibody and protein G beads (90-min rotation at 4°C). After three washings (IP buffer containing 0.3% octylglycoside), SDS sample buffer was added and the proteins were separated by SDS-PAGE (4%/10%) and immunoblotted for β1-integrin (MAB2252).

0019
For the luminescent immunoprecipitations, the beads were transferred into white microtiter plate wells (96-well) and treated with Rluc substrate (5 μg/ml coelenterazine [Nanolight Technologies]), and the luminescence was measured with a multilabel HTS counter (Victor2V; PerkinElmer).
0411
The cells were lysed (200 mM NaCl, 75 mM Tris, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% Triton-X-100, and Complete), and the amount of biotinylated integrin was assayed using the anti–β1-integrin antibody AIIB2 to capture the integrins and HRP anti-biotin antibody for ELISA detection.
0052
Ultrathin cruosections were prepared on a cryochamber (EM FCS; Leica), and thawed sections were incubated with a polyclonal antiserum raised against EGFP followed by incubation with protein A complexed to 5-nm gold particles according to standard procedures.
0019
HeLa cells were transiently transfected with GFP, GFP-Rab21, or GFP-Rab21GTP using Lipofectamine 2000 as described in the Immunoprecipitations section. 48 h after transfection, the cells were harvested and fractionated on a sucrose density gradient and analyzed by Western blotting as described previously (Hughes et al., 2002).
0432
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005).
0416
Green fluorescent cells were counted using a widefield epifluorescence microscope (narrow GFP filter and 20× objective).
0416
TIRFM was combined with conventional widefield epifluorescence microscopy and time-lapse series (frame rate ∼2/s) Widefield images were pseudocolored red and TIRFM images green.
0416
The Axioplan 2 microscope equipped with Plan-Apochromat 63× (NA 1.4) objective and a camera (Orca 2; Hamamatsu Photonics) was used for widefield epifluorescence time-lapse imaging at a rate of 2 frames/s.
0416
Fluorescence intensities for TIRFM and widefield epifluorescence microscopy were measured and analyzed with MetaMorph software.
0040
S3 shows the localization of organelle markers on the sucrose gradient–fractionated GFP-Rab21–expressing HeLa cells and immunogold electron micrographs of GFP-Rab21–positive structures in MDA-MB-231 cells.

0416
Video 2 shows a combined widefield epifluorescence and TIRFM analysis of MDA-MB-231 cells expressing GFP-Rab21, adhering to collagen. Video 3 shows a combined widefield epifluorescence and TIRFM analysis of MDA-MB-231 cells expressing GFP-Rab21GDP mutant, adhering to collagen.
0809
To investigate the mechanism of miRNA-mediated repression of mRNA translation and to determine the interactions of P-body components with the RNAi machinery, we constructed expression vectors for the yellow fluorescent protein (YFP)-tagged P-body proteins, Lsm1, RCK/p54, Dcp2, and eIF4E.

0019
These vectors were co-expressed in HeLa cells with Myc-tagged Ago2 and immunopurified using anti-Myc antibodies. The protein composition of isolated complexes was analyzed by immunoblot using antibodies against green fluorescent protein (GFP) or Myc. When total cell extracts (TCE) were analyzed to determine the protein expression efficiencies of the vectors used in these experiments (Figure 1A, TCE lane), all YFP- and Myc-tagged proteins were expressed. Analysis of immunopurified complexes revealed that Ago1, Dcp2, RCK/p54, and eIF4E formed complexes with Ago2 (Figure 1A, anti-Myc lane).
0019
To address this possibility, HeLa cells were transfected with vectors to co-express Myc-Ago2 and the YFP-tagged P-body proteins, Lsm1, RCK/p54, Dcp2, and eIF4E, subjected to RNase A digestion, and immunopurified. Analysis of immunopurified complexes showed that Ago1 and RCK/p54 interactions with Myc-Ago2 were not affected by RNase treatment, whereas the amounts of Dcp2 and eIF4E protein that co-purified with Myc-Ago2 decreased significantly (Figure 1A, anti-Myc lane). Control experiments analyzing Myc-Ago2 showed that equal amounts of complexes were purified (Figure 1A, anti-Myc lane).
0663
To examine the contents of these cytoplasmic foci, HeLa cells were transfected with expression vectors for YFP-Lsm1 and CFP-Ago2, or YFP-RCK/p54 and CFP-Ago2, and visualized 24 h later by confocal microscopy.
0012
To visualize protein–protein interactions in vivo, we used fluorescence resonance energy transfer (FRET) as a probe. In FRET, a fluorescent donor molecule transfers energy via a nonradiative dipole–dipole interaction to an acceptor molecule [46]. We used a well-known donor: acceptor fluorescent-protein pair, CFP:YFP, with a Förster distance (R 0) of 4.9 nm [47].

0019
In cells expressing CFP-Ago2 and YFP-Lsm1, FRET efficiency was not significant (1.62% ± 1.11%), corroborating our immunoprecipitation results (Figure 1A).
0019
Immunoblot analysis of affinity-purified miRISC using anti-Myc, anti-Flag, anti-RCK/p54, and anti-eIF4E antibodies (Figure 3C) showed that Myc-Ago1, Flag-Ago2, and RCK/p54 were associated with let-7 miRISC.
0019
24 h after transfecting cells with siRNA, real-time quantitative PCR showed that mRNA levels decreased by more than 90% and immunoblot analysis showed that RCK/p54 protein levels decreased significantly without affecting the levels of other P-body proteins including Lsm1 and Ago2 (Figure S2).
0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly complementary or mismatched sequence to the let-7 miRNA.
0052
HeLa cells were cultured in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2.
0019
To examine the RNA dependence of protein–protein interactions, TCEs (250 μg) were treated before immunoprecipitation with 0.2 μg/ul of RNase A for 20 min at room temperature. Myc-tagged proteins were precipitated by incubating overnight with polyclonal rabbit anti-Myc antibodies directly conjugated to agarose beads (Santa Cruz Biotech, California, United States). Samples were washed four times in lysis buffer and eluted by boiling for 5 min at 100 °C in SDS-PAGE sample loading buffer, separated by SDS-PAGE, and analyzed by immunoblot. For immunoblotting, antibodies included monoclonal mouse anti-GFP (BD Biosciences), anti-eIF4E and anti-Myc (Santa Cruz Biotech), anti-Flag (Sigma, St. Louis, Missouri, United States); polyclonal rabbit anti-Myc (Santa Cruz Biotech), anti-DDX6 (rck/p54; Bethyl Laboratories, Montgomery, Texas, United States); and polyclonal chicken anti-Lsm1 (GenWay Biotech Incorporated, San Diego, California, United States).
0663
The signals were detected by a Leica (Wetzlar, Germany) confocal imaging spectrophotometer system (TCS-SP2) attached to a Leica DMIRE inverted fluorescence microscope equipped with an argon laser, two HeNe lasers, an acousto-optic tunable filter (AOTF) to attenuate individual visible laser lines, and a tunable acousto-optical beam splitter (AOBS).

0055
For FRET studies, HeLa cells co-expressing CFP- and YFP-tagged proteins were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed three times with PBS.

0663
After the final wash, cells were visualized with a Leica confocal imaging system as described above.

0055
FRET experiments were performed by an acceptor photobleaching method as described [48,49,68]. FRET efficiencies were measured and images were analyzed using Leica confocal software.

0663
After the final wash, samples were counterstained with Hoechst 33258 to visualize nuclei with a Leica confocal imaging system as described above.
0019
To quantify RNAi effects, cell lysates were prepared from siRNA-treated cells 24 h post-transfection.

0420
Total cell lysate (150 μg in 200 μl of reporter lysis buffer) was measured using a Safire plate reader (TECAN).

0019
GFP fluorescence was detected in cell lysates by exciting at 488 nm and recording emissions from 504–514 nm.

0019
RFP fluorescence was detected in the same cell lysates by exciting at 558 nm and recording emissions from 578–588 nm.
0019
Cell lysates (50 μl) were incubated with 10 μl of 100% TCA on ice for 30 min, and the protein precipitate was collected on GF/C filter paper (Whatman, Clifton, New Jersey, United States), washed with 5 ml of 95% ethanol and counted in scintillation fluid.
0663
HeLa cells expressing YFP-Lsm1 and CFP-Ago2 (a, b, and c), YFP-RCK/p54 and CFP-Ago2 (d, e, and f) were visualized by confocal microscopy at 24 h post-transfection.
0055
(D) FRET efficiencies between different P-body protein donor: acceptor pairs. HeLa cells co-expressing YFP-RCK/p54 and CFP-Ago2, YFP-Lsm1 and CFP-Ago2, YFP-RCK/p54 and CFP, YFP-Ago1 and CFP-Ago2, YFP-Ago2 and CFP-Ago1, YFP-RCK/p54 and CFP-Ago-1, as well as YFP-Ago1 and CFP, were fixed and FRET efficiencies were measured.
0019
Active human RISC from HeLa cells expressing Flag-Ago1 was captured by biotin-siRNA and its protein composition was analyzed by immunoblot using anti-Flag, anti-Ago2, anti-RCK/p54, anti-Lsm1, and anti-eIF4E antibodies.
0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly complementary or mismatched sequence to the let-7 miRNA.
0019
Supernatant (S) and beads (B) after biotin capture were analyzed by immunoblot using anti-Myc, anti-Flag, anti-RCK/p54, and anti-eIF4E antibodies.
0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly matched or a mismatched sequence to the let-7 miRNA.
0019
Western-blot analysis of whole cell lysates indicated that both proteins were expressed, albeit at different levels, and that expression of ephrin-B1 did not influence the phosphorylation status of Cx43 (detected by differences in mobility on a SDS-PAGE), which is known to regulate GJC (Figure 7A).
0052
Trypsin was inactivated by addition of complete medium (DMEM containing 15% FCS) and cells were dissociated by trituration with a glass Pasteur pipette.
0052
Primary NCCs were isolated from dissected branchial arches of E9.5 embryos as described above, except cells were cultured in F12 medium supplemented with 10% FCS.
0019
Protein lysates from NIH 3T3 cells expressing ephrin-B1 were incubated with ephrinB1-Fc, and the presence of Cx43 in the affinity complexes was assessed by Western blot (b) Cx43 was immunoprecipitated from NIH 3T3 cells expressing ephrin-B1 and the presence of ephrin-B1 in the immunocomplexes was detected by Western blot (right panel).
0019
IP, immunoprecipitation; Wcl, whole cell lysate.
0019
Whole cell lysates (a) and affinity precipitations (b) were analyzed by Western blot.
0417
Although the details of how replication occurs on chromatin are poorly understood, it is possible that remodeling activities that promote protein-protein and protein-DNA interactions on chromatin are important to allow efficient replication of these templates [11-13].
0054
Cell cycle progression profiles of Δabp1, cdc23-M36 and cdc23-M36 Δabp1 at 30°C (from 1–6 hrs) and 25°C at time zero, by flow cytometry anaylsis.
0054
We analyzed DNA content by flow cytometry to determine the precise arrest points for the different strains (Figure 3B).
0054
FACS analysis (DNA content) for Δabp1 strain (wild-type (wt) as control).
0018
First, Abp1 not only interacts with Cdc23 (Mcm10) protein in the two-hybrid analysis, but deletion of Abp1 lowers the restrictive temperature for cdc23-M36, consistent with its proposed role in DNA replication.
0054
Our flow cytometry analysis suggests that cdc23+ (MCM10) is required for DNA replication initiation.
0054
Cells were then returned to the permissive temperature of 25°C, collected at the indicated times and fixed for flow cytometry analysis (FACS).

0054
Cells were then collected at the indicated times and prepared for FACS analysis.
0226
Recombinant purified His-tagged Bcl-xL was exposed to alkaline conditions to cause partial deamidation and separated by anion-exchange chromatography into three peaks (Figure 2C, peaks A, B and C).
0054
Figure 5C and Figure S6B illustrate that double staining for Annexin V and propidium iodide (PI) followed by FACS analysis revealed a major increase in Annexin V+ PI− (apoptotic) cells following transduction with the negative control shRNA followed by either γ irradiation or treatment with etoposide, whereas there was no increase in apoptotic cells above baseline in the cells depleted of NHE-1: DNA damage–induced apoptosis was blocked 100%. Comparable results were obtained by measuring the sub-G1 peak by FACS (unpublished data) and NHE-1 depletion also correlated with increased survival (Figure S5B).
0114
Furthermore, comparison of the crystal structures of native rat Bcl-xL with its deamidated version has revealed significant differences [10]; the structural implications of introducing iso-Asp residues into the disordered loop environment of Asn52/Asn66 merits further work.
0071
Aliquots of the digestion mixtures were analysed by liquid chromatography mass spectrometry (LC-MS) on a quadrupole TOF mass spectrometer (Qstar pulsar i, Applied Biosystems-MDS Sciex), with online separation by reversed-phase nano-LC.
0019
Cell lysates were resolved by standard Laemmli's SDS-PAGE (pH 8.8) unless otherwise stated. For immunoprecipitations: rat Bim antibody (Oncogene, San Diego, California, United States) was coated to goat-anti-rat immunoglobulin-agarose; rabbit Puma antibody was coated to protein A-sepharose; mouse NHE-1 antibody was coated to protein G-sepharose; rabbit Bcl-xL antibody was coated to goat-anti-rabbit immunoglobulin-agarose. Lysates were precleared with the appropriate agarose.
0054
Intracellular pH was measured using a standard ratiometric method with a pH-sensitive fluorophore SNARF-1 by flow cytometry [44].

0054
FACS data were analysed using Flowjo software to obtain the ratio based on the Fl3/Fl2 channels.
0054
Cells were stained with 20 μg/ml PI (with 50 μg/ml RNase A) and analysed by flow cytometry, gating on the CD4−CD8− subset as necessary.
0054
GFP-positive cells were sorted by flow cytometry using a FACsAria.
0054
Cells positive for both GFP and DsRed were sorted by flow cytometry and used for subsequent experiments.
0054
The purity of PBMCs was routinely checked by staining with antibodies CD3-Cy5, CD19-Fitc, and B220-PE and was analysed by flow cytometry.
0054
(A) Wild-type thymocytes were pre-incubated with or without Z-VAD-fmk (200 μM), and were then cultured with or without etoposide for 24 h, harvested, and apoptosis was measured by measuring the sub-G1 peak by flow cytometry.
0054
48 h later, GFP+ DsRed+ cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture.
0226
(C) Anion exchange chromatography of purified rBcl-xL.
0226
The three different forms of Bcl-xL (A, B, and C) purified by anion-exchange column chromatography shown in (C) were incubated in wild-type thymic lysates (1.5 × 107 cell equivalents) at 4 °C for 2 h and then precipitated using nickel beads.
0054
pHi was measured using SNARF by FACS in the gated live CD4−CD8− subset.
0052
Wild-type thymocytes were maintained in RPMI-1640/10% bovine fetal calf serum buffered at the indicated pH with Tris-HCl for 20 h in the presence of 20 μM monensin prior to lysis and immunoblotting for Bcl-xL.
0054
(E) Aliquots of cells used in (D) were assessed for pHi by FACS.
0054
(F) Apoptosis of aliquots of the cells from (D) was analysed by FACS.
0054
GFP-positive cells were FACS sorted (left panel) and cultured in media with the pHe shown for 24 h or 48 h, then processed for immunoblotting with Bcl-xL antibody (middle panel).
0019
Cell lysates were processed by immunoblotting for Bcl-xL or β-actin (loading control).
0054
The lower left FACS histogram shows the infection efficiency for nontransfected (non), empty-vector transfected (vector), or NHE-1 transfected (NHE-1) cells as percentage GFP-positive cells.
0054
pHi was measured by FACS on live CD4−CD8− cells, and the sub-G1 peak was analysed by FACS on CD4−CD8− cells to assess apoptosis.
0054
(C) Aliquots of the cells from (A) were analysed for apoptosis by Annexin V/PI staining using flow cytometry, as illustrated in a representative experiment (total n = 5).
0054
Patients' cells (PBMC, in the range 85%–95% CD19+B220+) were incubated at pHe values of 7.2, 8.0, or 8.5, and the pHi values were monitored by SNARF-1 staining using flow cytometry. Apoptosis was evaluated by measurement of sub-G1 peaks using flow cytometry.
0052
The same cell aliquots cultured in RPMI/10% FCS for 24 h or 48 h were analysed for apoptosis by sub-G1 staining (right panel).
0014
Here, we demonstrate that β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously.
0014
Even though these highly homologous receptors both activate the G protein stimulatory for adenylyl cyclase (Gs), signaling through β1AR and β2AR produces clearly distinguishable biological effects (Xiang and Kobilka, 2003; Xiao et al, 2004).
0014
One of the emerging mechanisms that safeguard the specificity of G-protein-coupled receptor/cAMP signaling is the control of cAMP transients via degradation by cyclic nucleotide phosphodiesterases (PDEs) (Conti and Beavo, 2007). Biochemical, electrophysiological, and in vivo imaging studies are consolidating the idea that occupancy of different receptors generates a nonuniform pattern of activation of cAMP effector proteins such as PKA (cAMP-dependent protein kinase). PDEs play a critical role for the specificity in cAMP-signaling by preventing the free diffusion of cAMP, thus, effectively creating cyclic nucleotide microdomains and/or cAMP gradients that can be sensed by the cell (Zaccolo and Pozzan, 2002; Xiang et al, 2005; Fischmeister et al, 2006).
0019
To probe for a possible signaling complex including the β1AR and a PDE, mouse neonatal cardiomyocytes were infected with an adenovirus encoding a Flag-tagged β1AR, and the receptor was subsequently immunoprecipitated using an antibody against the tag. A significant amount of endogenous PDE activity was recovered in the β1AR immunoprecipitation (IP) pellet (Figure 1A).

0019
Whereas ablation of PDE4A or PDE4B had no effect, inactivation of the PDE4D gene prevented co-IP of PDE activity with the β1AR (Figure 1B).

0019
This conclusion is further supported by western blot analysis of the immunoprecipitated PDE.
0014
Thus, the concentration-dependence of dissociation of the β1AR/PDE4D complex is comparable to that of receptor occupancy by NorEpi rather than that of receptor-induced cAMP production, which is in the nanomolar range.
0012
The β1AR/PDE4D complex is present in the absence of agonist and dissociates after receptor occupancy, whereas agonist binding to the β2AR is a prerequisite for the recruitment of the β-arrestin/PDE4D complex to the receptor. Thus, under basal conditions, PDE4D is poised to control local cAMP concentration and PKA activity in the vicinity of the β1AR (see Figure 6), whereas it affects β2AR signaling only after ligand binding and β-arrestin recruitment.
0019
Recombinant Flag-tagged βARs and Myc-tagged PDE4D splice variants were detected in western blots using antibodies against their respective tags (mouse monoclonal α-Flag AB, Sigma Aldrich; mouse monoclonal α-Myc AB, Roche Applied Sciences).
0052
They were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Nu Serum IV (BD Falcon), 5% fetal bovine serum (FBS), 1 mM glutamine, 20 μg/ml gentamycin, and 1 × ITS media supplement (Sigma) on plates precoated with 10 μg/ml laminin.

0052
HEK293 and MEF cells were cultured in DMEM supplemented with 10% FBS, 1 mM glutamine, 30 μg/ml penicillin, and 100 μg/ml streptomycin.
0019
Flag-tagged receptors were then immunoprecipitated using M1-affinity resin (α-Flag antibody resin; Sigma Aldrich).
0019
Flag-tagged β1AR and β2AR were also expressed in Sf9 cells and subsequently purified in a two-step procedure consisting of an initial affinity chromatography using M1-resin (immobilized anti-Flag antibody; Sigma Aldrich), followed by an alprenolol-sepharose affinity column.
0019
for 30 min), and soluble extracts were immunoprecipitated using 30 μl ProteinG Sepharose and the respective PDE4 subtype, or PDE4D splice variant antibodies, as well as IgG as a control. After incubation for 2 h at 4°C, the resin was washed three times, and PDE recovered in the pellet was detected by PDE activity assay or western blotting.
0019
(B) Co-IP of β1AR and PDE activity from cardiomyocytes deficient in PDE4A, PDE4B, or PDE4D, and wild-type controls.
0019
(A, B) Co-IP of exogenous β1AR and Myc-tagged PDE4D splice variants expressed in HEK293 cells.

0019
(C) Shown is the co-IP of exogenous β1AR and PDE4D8-Myc from extracts of MEFs derived from mice deficient in β-arrestin 1 and 2 (βarr1/2KO) or from wild-type controls (WT-MEF). (D, E) PDE4D3, and Flag-tagged receptors, β1AR and β2AR, were affinity purified after baculovirus expression (see Supplementary Figure 1). Purified PDE and (βARs) were then combined and the βARs immunoprecipitated.
0019
At the end of incubation, cells were lysed, PDE4D5, 8, and 9 were immunoprecipitated with variant-specific antibodies, and the PDE activity recovered in the IP pellet was measured.
0019
(A, B) Neonatal cardiac myocytes expressing a Flag-tagged β1AR were treated for 3 min with 100 nM Norepinephrine before cell lysis and IP with M1 (α-Flag) resin.

0019
(E, F) Neonatal cardiac myocytes coexpressing a Flag-tagged β1AR and either GFP, a catalytically inactive PDE4D8 construct (PDE4D-DN; see also Supplementary Figure 5), or a catalytically inactive PDE3A1 (PDE3A1-DN) were subjected to α-Flag(M1)-IP, and the phosphorylation of the β1AR was subsequently detected in IB using a PKA-substrate-specific antibody.
0114
We generated a model of AIRE–PHD1 complexed with the H3K4me0 peptide on the basis of the crystal structure of the BPTF–PHD finger bound to H3K4me3 and performed molecular dynamics calculations for 10 ns.
0077
Remarkably, residues G305, G306 and G313 showed strong shifts when bound to H3K4me2 and disappeared completely from the NMR spectrum owing to line-shape broadening on binding to H3K4me3, indicating an involvement of this region in peptide binding.
0065
NMR binding, fluorescence titration assays and isothermal titration calorimetry thermodynamic analysis.

0077
NMR binding, fluorescence titration assays and isothermal titration calorimetry thermodynamic analysis. Details on NMR titrations, fluorescence spectroscopy and thermodynamic measurements are described in the supplementary information online.
0402
Cell lines, expression analysis and chromatin immunoprecipitation.

0402
DNA ChIP was performed essentially according to Upstate Chromatin Immunoprecipitation Assay protocol.
0402
DNA ChIP with (B) anti-AIRE, (C) anti-H3K4me3 and (D) anti-H3. The fold differences are normalized to input fractions and shown in comparison with the background level (ChIP with IgG from HEK-control cells (=1)) of each primer set.

0402
AIRE, autoimmune regulator; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; H3K4me3, histone H3 trimethylated at lysine 4.
0108
In addition, replication of single-stranded DNA of M13 phage was normal in Plx1-depleted extracts, indicating that DNA elongation was not affected by Plx1 deficiency (Supplementary Figure 3).
0858
Interphase extracts (500 μl) were incubated three times for 40 min with 100 μl of protein A-Sepharose beads, 4B fast flow (Sigma) coupled to 200 μl of anti-Plx1 serum, 100 μl of anti-Mcm3 or 200 μl of anti-Tipin serum at 4°C for 2 h in the presence of 1 mM AEBSF (4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride).
0108
Replication of single-stranded DNA of M13 phage was performed in a similar manner using 12.5 ng/μl of DNA.
0858
(A) Immunodepletion of Plx1.
0077
To obtain the p62 PH-D-bound structure of hTFIIEα AC-D using NMR, we performed NMR titration experiments in buffers both with and without 100 mM NaCl for both domains (Supplementary Figures 1–3). Although in the 100 mM NaCl buffer the dissociation constant (Kd) between AC-D and p62 PH-D was estimated from the titration plots as 376±81 nM (Supplementary Figure 1C) or 237±82 nM (Supplementary Figure 2C), the NaCl-free buffer NMR titration experiment showed much stronger binding affinity between AC-D and p62 PH-D because of the slow exchange timescale with a Kd below about 150 nM (Supplementary Figure 3).
0077
In the binding of VP16 TAD to Tfb1 PH-D, the Kd value estimated by NMR titration experiment was ∼4000–7000 nM (Di Lello et al, 2005).
0077
Measurements of NMR spectra, structural calculations and NMR titration experiments are described in Supplementary data.
0019
The bound proteins were released by boiling in SDS–PAGE loading buffer, separated by SDS–PAGE and detected by western blotting with anti-hTFIIEα rabbit antiserum (1:3000 dilution), anti-FLAG M2 monoclonal antibody (Sigma) and anti-p53 (DO-1) (Santa Cruz) using the enhanced chemiluminescence detection system (GE Healthcare).
0077
Left, superposition of the backbone heavy atoms of the 20 lowest energy NMR structures.
0077
(A) Superposition of the backbone heavy atoms of the 20 lowest energy NMR structures.
0019
Bound mutants were subjected to SDS–PAGE and detected by western blotting with anti-FLAG antibody.

0019
Bound proteins were subjected to SDS–PAGE and detected by western blotting with anti-FLAG, anti-p53 and anti-hTFIIEα antisera.
0052
RPMI 1640 medium was purchased from Invitrogen (Gaithersburg, MD). Fetal bovine serum (FBS) and charcoal/dextran-treated FBS were purchased from HyClone Laboratories (Logan, UT).
0052
The human breast cancer cell lines MCF7 and T47D, and cervical carcinoma cell line Hela were obtained from ATCC (Rockville, MD) and maintained in RPMI-1640 medium supplemented with 10% FBS (complete medium) at 37°C in 5% CO2.

0052
Transient transfection experiments were performed in RPMI-1640 medium supplemented with 10% charcoal/dextran-treated FBS (stripped medium).
0420
After removal of the residual dye and medium, 100 ul of dimethylsulfoxide was added to each well, and the absorbance at 570 nm was measured using BMG microplate Reader (BMG Labtech, Inc., Durham, NC).
0096
KL generated GST fusion protein for pull down analysis.
0071
Membrane-bound proteins were separated from soluble proteins by Sepharose CL-2B gel filtration chromatography.
0071
In incubations of 20 nM tBid and liposomes, tBid bound effectively to liposomes as assayed by gel filtration chromatography (Figure 1B).
0019
Unlike the conformational change that accompanies tBid-induced insertion of Bax into membranes, the liposome-induced conformational change also disappears if liposomes are solubilized in CHAPS prior to immunoprecipitation (Figure 5A, compare lanes 1 and 3).
0019
OSM-11 was not detected in cell lysates (unpublished data).
0432
DSL-1, OSM-11, LAG-2 extracellular domain (LAG-2Ex), EGL-17, or LIN-3 was fused to the GAL4 DNA binding domain (DB); the first six LIN-12 EGF repeats were fused to the GAL4 activation domain (AD).
0416
Pn.p cells and descendents were identified by differential interference contrast (DIC) imaging on a Zeiss Axioskop2.
0114
We initially attempted to solve the crystal structure of full-length human α-parvin, but no crystals could be obtained. However, limited proteolysis led to the identification of a stable fragment, α-parvin-CHC, that readily crystallized.

0114
The crystal structure of α-parvin-CHC at 1.05 Å resolution was determined by molecular replacement using an ensemble of homologous type-1 CH domain structures as search model.

0114
The refined structural model includes α-parvin residues 246–372 (molecule A) or 247–372 (molecule B) and represents the first high-resolution crystal structure of a type-5 CH domain (Figure 1B).
0114
Surprisingly, however, the orientation of LD1 is reversed compared to LD2 and LD4 in the corresponding crystal structures.
0077
This phenomenon is also illustrated in Figure 5, using two diagnostic NMR signals as examples: the resonance originating from residue 257 is predicted to experience strong PRE in the forward mode (calculated distance from spin-label 10.7 Å) but should only be weakly affected in the backward mode (calculated distance 20.2 Å).
0114
We thus conclude that the crystal structures of the α-parvin-CHC/LD complexes along with the solution NMR studies presented here provide a relevant description of LD recognition by α-parvin.
0077
Small changes in a variety of factors, such as experimental conditions or the length of the paxillin fragment studied, might influence the observed binding orientation in NMR, where a mean is detected.
0114
Interestingly, the crystal structure of α−parvin-CHC presented here shows that part of the putative ABS2 region is obstructed by the N-linker helix.
0071
On the basis of gel filtration experiments with the N-terminal CH domain of α-parvin, Wang et al.
0071
After cleavage with recombinant human rhinovirus 3C-protease, the sample was subjected to size exclusion chromatography (Superdex 75, GE Healthcare) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, and 2 mM EDTA.
0226
After elution with 200 mM potassium phosphate, 10 mM NaCl, 4 mM DTT, and 2.5% glycerol, the protein was dialyzed into 25 mM potassium phosphate (pH 8.0), 1.5 mM NaCl, 2 mM DTT, and 2.5% (v/v) glycerol for subsequent anion exchange chromatography (MonoQ, GE Healthcare) and gradient elution with 250 mM potassium phosphate (pH 8.0), 15 mM NaCl, 250 mM KCl, 2 mM DTT, and 2.5% glycerol.
0053
For fluorescence anisotropy studies, LD1 with 5-carboxyfluorescein (5-FAM) attached to the ɛ-amino group of the C-terminal lysine was used.
0114
Structures of α-parvin-CHC/LD complexes were solved by molecular replacement with the α-parvin-CHC apo structure using PHASER (McCoy, 2007), and refined with REFMAC5, allowing one TLS group per polypeptide chain.
0077
For PRE measurements, 1H-15N HSQC experiments were recorded on 230 μM 15N-enriched α-parvin-CHC in the presence of 250 μM PROXYL-labeled LD1 peptide in 50 mM sodium phosphate and 100 mM NaCl (pH 6.9) in the absence and presence of 5 mM ascorbate at 25°C.

0114
For the simulation of PRE effects in both unidirectional binding modes, the crystal structures of α-parvin-CHC bound to LD1 and LD4 were protonated with the program PDB 2PQR (Dolinsky et al., 2004).
0077
NMR Titrations of α-Parvin-CHC with Paxillin LD Peptides
0077
(A) Details of the 1H-15N HSQC spectra of 230 μM 15N-enriched α-parvin-CHC and 250 μM PROXYL-labeled LD1 peptide in the absence (left) and presence (right) of 5 mM ascorbate.

0114
The binding orientation of LD1 seen in the crystal structure is denoted “forward.”.
0114
Paxillin residues ordered in the crystal structures of the α-parvin-CHC/LD complexes are underlined and those forming contacts with α-parvin-CHC within a radius of 4 Å are boxed.
0019
A single round of transcription on the HIS4 template was allowed, and RNApII was immunoprecipitated from the supernatant through the Rpb3 subunit (Figure 5E).
0402
We note that in some ctk1Δ strain backgrounds, the abnormal cross-linking of basal factors observed in our ChIP analyses was not as pronounced.
0019
For Rpb1 (BWG16 Covance), Sua7, TBP, Kin28, Tfb1, Tfa2, and Rap1 (y-300, Santa Cruz) immunoprecipitations, the antibodies were preincubated with protein A-sepharose CL-4B (Amersham) for 1 h at room temperature and then incubated with chromatin solution overnight at 4°C. For Rpb3 immunoprecipitation, antibody (1Y26, NeoClone) was preincubated with protein G-sepharose (Amersham).

0019
The signal for each specific gene primer in the immunoprecipitation was then divided by this ratio to convert the signal to normalized units. This value was divided by the immunoprecipitation signal of the non-transcribed control product to determine the fold enrichment of the ChIP over background signals.
0276
Beads were washed five times with buffer E and loaded onto SDS–PAGE gels together with 50 mg of input material.
0019
The abnormal cross-linking of basal transcription factors in ctk1Δ cells is confirmed by independent immunoprecipitation of TAP-tagged TFIIF subunits.

0019
IgG agarose was used for immunoprecipitation of TAP-tagged proteins.
0402
Cells with kin28-T17D (YSB592) or srb10-D290A (YF243) alleles were grown at 30°C and prepared for ChIP analysis.

0019
Chromatin from each strain was immunoprecipitated with anti-TBP.

0402
ChIP was carried out in the indicated strains.

0402
Each strain was then transformed with RNA14–TAP (creating YSB1188, YSB1189, and YSB1190, respectively) before ChIP analysis.

0019
Protein A- or protein G-sepharose was used for TBP or Rpb3 immunoprecipitation and rabbit IgG agarose was used for RNA14–TAP pull-down.

0096
Protein A- or protein G-sepharose was used for TBP or Rpb3 immunoprecipitation and rabbit IgG agarose was used for RNA14–TAP pull-down.

0402
Cells were grown at 22°C, shifted to 37°C (non-permissive temperature) for 4 h as indicated, and prepared for ChIP analysis. (E) The Ctk1-deficient strain (YSB854) was transformed with either of the plasmids YCplac22 (empty vector), YCplac22-CTK1HA (T338A), YCplac22-CTK1-DN, or pRS316-CTK1 and used for ChIP analysis.
0019
To measure the association between RNApII and the basal transcription factors after termination, the supernatant was removed after incubation with NTPs for 2 min and immunoprecipitated with anti-Rpb3 antibody. (F) Coimmunoprecipitated proteins with Rpb3 from WT and ctk1Δ cells were determined by western blot using antibodies against non-scaffold (Tfg2 or Sua7) and scaffold components (TBP, Tfb1, Kin28, or Tfa2).
