0096
To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK.
0423
We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro.
0004
The glutathione S-transferase (GST)-Bcl-GL recombinant protein was expressed in Escherichia coli strain BL21 codon-plus RIL competent cells (Stratagene). Purification of the recombinant proteins was performed using Glutathione Sepharose 4B beads (GE Healthcare) under nondenaturing conditions according to the supplier's instructions. For confirmation of direct binding of BCL-GL and MELK, we removed GST from GST-fused BCL-GL protein using PreScission protease (GE Healthcare) according to the supplier's instructions.
0007
HeLa cells were transiently co-transfected for 48 hours with 8 μg of plasmid constructs encoding Flag-tagged full-length Bcl-GL or a series of Flag-tagged partial Bcl-GL proteins (FL, N-1, N-2, N-3, N-4, C-1, C-2 and C-3) as well as the same amount of plasmid encoding HA-tagged WT-MELK, using the FuGENE6 transfection reagent (Roche). Cells were lysed with lysis buffer as described above. The lysates were pre-cleaned with normal mouse IgG (1.2 μg) and rec-Protein G Sepharose 4B (Zymed, San Francisco, CA, USA) at 4°C for 30 minutes. Subsequently, the lysate was incubated with anti-Flag agarose M2 gel (Sigma-Aldrich) at 4°C for 12 hours. After washing three times with lysis buffer, proteins on beads were eluted with SDS sample buffer.
0096
To investigate the biological functions of MELK in breast cancer cells, we searched for substrates of MELK in cancer cells by in vitro protein pull-down assays using wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK) recombinant proteins. Comparison of silver staining of SDS-PAGE gels containing the pulled-down proteins identified an approximately 30 kDa protein in the lane corresponding to proteins pulled-down with WT-MELK but not in that corresponding to proteins pulled-down with D150A-MELK (Figure 3a).
0007
To validate an interaction between WT-MELK and Bcl-GL, we constructed plasmids designed to express HA-tagged WT-MELK (HA-WT-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL). These plasmids were co-transfected into HeLa cells and the proteins immunoprecipitated with anti-Flag antibody. Immunoblotting of the precipitates using anti-HA antibodies indicated that Flag-Bcl-GL was co-precipitated with HA-WT-MELK (Figure 3c).
0096
Furthermore, we demonstrated that His-tagged WT-MELK could pull-down with Bcl-GL but His-tagged D150A-MELK could not, indicating that, in vitro, Bcl-GL interacts directly with WT-MELK but not with D150A-MELK (Figure 3d).
0007
To further determine which segment of Bcl-GL can interact with WT-MELK, we performed co-immunoprecipitation analyses using HA-WT-MELK and partial Bcl-GL proteins tagged with Flag (Figure 3e). After co-transfection of plasmid clones into HeLa cells, we performed immunoprecipitation with an anti-Flag antibody, and then immunoblotting with an anti-HA antibody.
0096
Thus, to investigate the biological significance of MELK in breast cancer cells, we searched for a possible substrate(s) of MELK by means of in vitro pull-down assays with recombinant wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK).
0007
(c) Interaction of MELK with Bcl-GL. Extracts from HeLa-cells transfected with HA (hemagglutinin)-tagged WT-MELK (HA-WT-MELK) or Flag-tagged Bcl-GL (Flag-Bcl-GL), or a combination of these, were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with an anti-HA antibody.
0007
(f) Determination of the WT-MELK binding regions of Bcl-GL by immunoprecipitation. The HA-tagged WT-MELK and various peptide sequences of Flag-tagged Bcl-GL (Figure 3e) were pulled down by immunoprecipitation with Flag-M2 antibody and then immunoblotted with rabbit anti-Flag antibody. The expression of HA-tagged WT-MELK in total cell lysates was confirmed by western blotting analysis. As a control, immunoprecipitation was performed from cells co-transfected with pCAGGSn3FC (Mock) and HA-tagged WT-MELK (HA-WT-MELK) through all steps.
0006
To explore the potential interplay between Trk receptors and Cdk5, we first examined if Trk receptors associated with Cdk5 or p35. TrkA, TrkB, or TrkC was overexpressed together with Cdk5 or p35 in COS7 cells, and immunoprecipitation was performed with Cdk5, p35, or pan-Trk antibody. Interestingly, all three Trk receptors were observed to associate with Cdk5 (Figure 1B) and p35 (Figure 1C), while no association was observed when immunoprecipitation was performed with IgG control. Since both TrkB and its ligand BDNF are abundantly expressed in the brain throughout development, we next proceeded to verify the interaction between TrkB and Cdk5/p35 in postnatal brains. We found that TrkB associated with both p35 and Cdk5 in postnatal day 7 (P7) rat brain lysates (Figure 1D).
0006
TrkA, TrkB, and TrkC were overexpressed in COS7 cells and immunoprecipitated by pan-Trk antibody. Incubation with Cdk5/p25 revealed that TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25 in vitro (Figure 2A). This is in agreement with the lack of Cdk5 consensus sites in TrkA, and points to the possibility that Cdk5 may phosphorylate TrkB and TrkC at the Cdk5 consensus sites at the juxtamembrane region (Figure 1A). To examine this possibility, a GST fusion protein containing only the juxtamembrane region of TrkB was prepared.
0419
Rho GTPases, including RhoA, Rac1, and Cdc42, are key regulators of actin cytoskeleton dynamics. Since BDNF stimulation has been observed to activate Rac1 and Cdc42 in neurons [15], we were interested to delineate if Rho GTPases contribute to BDNF-stimulated dendritic growth. To investigate if Rho GTPases are involved, and to identify the Rho GTPase(s) implicated, hippocampal neurons were transfected with WT or DN Rac1, Cdc42, or RhoA. We found that while overexpression of WT and DN Rac1 increased the basal number of dendrites in the absence of BDNF treatment, overexpression of both forms of Rac1 abolished BDNF-stimulated dendritic growth. On the other hand, while overexpression of DN RhoA slightly enhanced primary dendrites irrespective of BDNF stimulation, overexpression of both WT and DN forms of RhoA inhibited BDNF-stimulated dendritic growth. Remarkably, in contrast to the inhibition of BDNF-stimulated dendritic growth in cells overexpressing WT Rac1 and RhoA, BDNF stimulation of hippocampal neurons overexpressing WT Cdc42 resulted in an increase in primary dendrites, which was nearly abolished by overexpression of DN Cdc42 (Figure 6A). Our observations therefore suggest that while Rac1 and RhoA may also modulate BDNF-stimulated dendritic growth, it is the activation of Cdc42 following BDNF stimulation that most likely mediates the increase in primary dendrites by BDNF.
0419
In the current study, we demonstrated that Ser478 phosphorylation of TrkB by Cdk5 is essential for the Cdc42-dependent increase in primary dendrites triggered by BDNF, thus adding a new regulatory component to the mechanisms involved in Rho GTPase activation by neurotrophin. Although the precise downstream pathways by which this phosphorylation affects Cdc42 activation remains to be determined, our observations provide some interesting insights.
0006
For immunoprecipitation, 1–2 mg of protein lysates was incubated with 1 μg of the corresponding antibody at 4 °C overnight with rotation. Forty microliters of protein G Sepharose (Amersham Biosciences) pre-washed with 1× PBS was added and rotated at 4 °C for 1 h. After intense washing with the lysis buffer, the immunoprecipitated protein and its associated proteins were analyzed by SDS-PAGE and Western blotting.
0006
(B) Cell lysates from HEK293T cells overexpressing Cdk5 and TrkA, TrkB, or TrkC were immunoprecipitated (IP) with Cdk5 antibody and immunoblotted with pan-Trk antibody. TrkA, TrkB, and TrkC were all observed to associate with Cdk5.
0006
(C) Cell lysates from HEK293T cells overexpressing p35 and TrkA, TrkB, or TrkC were immunoprecipitated with p35 antibody and immunoblotted with pan-Trk antibody. TrkA, TrkB, and TrkC were all observed to associate with p35.
0006
(D) Brain lysate from P7 rat brain was immunoprecipitated with pan-Trk, p35, or Cdk5 antibody and immunoblotted with p35, Cdk5, and TrkB antibodies. Rabbit normal IgG was used as a control. TrkB was observed to associate with both p35 and Cdk5 in P7 rat brain.
0096
(E) The membrane fraction of adult brain lysates was incubated with or without Flag-tagged Cdk5. Flag-tagged Cdk5 pulled down TrkB from the membrane fraction of adult brain lysates.
0006
(F) Brain lysates from P7 p35+/+ or p35−/− mouse brains were immunoprecipitated with p35 and Cdk5 antibodies and immunoblotted with p35, Cdk5, and TrkB antibodies. Rabbit normal IgG served as a control. Association between Cdk5 and TrkB was abolished in p35−/− brain, indicating that p35 was required for the association between Cdk5 and TrkB.
0006
(A) Lysates from COS7 cells overexpressing TrkA, TrkB, and TrkC were immunoprecipitated with pan-Trk antibody and incubated with Cdk5/p25 in an in vitro kinase assay. TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25.
0096
Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.
0006
(E) Full-length TrkB WT, M1, M2, and DM were overexpressed with or without Cdk5/p35 in HEK293T cells. In the absence of Cdk5/p35, Ser478-phosphorylated TrkB (p-Ser TrkB) was not detected. Overexpression of Cdk5/p35 resulted in phosphorylation of TrkB WT at Ser478, but phosphorylation at Ser478 was essentially abolished when TrkB M1 and DM were overexpressed. IP, immunoprecipitation.
0006
(A) Cortical neurons were stimulated with BDNF for different time intervals. Lysates were immunoprecipitated (IP) with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. BDNF stimulation for 15 min resulted in a marked increase in Cdk5 activity in cortical neurons. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.
0006
(B) Addition of Trk inhibitor K252a abolished BDNF-induced increase in Cdk5 activity. Cortical neurons were pretreated with vehicle control (DMSO) or K252a for 30 min before stimulation with BDNF for 15 min. Lysates were immunoprecipitated with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. We found that K252a pretreatment markedly reduced the increase in Cdk5 activity triggered by BDNF stimulation, indicating that the induction of Cdk5 activity was dependent on TrkB activation. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation in the presence or absence of K252a treatment was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.
0006
(C) Cortical neurons were treated with BDNF for 20 min. Lysates were immunoprecipitated with p35 antibody and immunoblotted with TrkB, p35, or Cdk5 antibody. While association between Cdk5 and p35 was not affected by BDNF stimulation, association between p35 and TrkB increased following 20 min of BDNF stimulation.
0096
GST-pull down and co-immunoprecipitation assays were performed to further characterize this interaction.
0006
GST-pull down and co-immunoprecipitation assays were performed to further characterize this interaction.
0006
Coimmunoprecipitation of BRCA1 and PP1. HEK293T kidney cells were transfected with vectors encoding untagged BRCA1 under the control of a CMV promoter, and vectors encoding Flag-PP1α, β, γ or Flag-Laf4. (A) A western blot probed with BRCA1 shows that immunoprecipitation of protein with an antibody against the Flag-PP1α, β or γ proteins, but not Laf4, co-immunoprecipitates BRCA1 (Lanes 1A-1D). It should be noted that the band observed slightly lower than BRCA1 in lane 1D is a non-specific background band. Lanes 1E-1H show immunoprecipitation of BRCA1 using antibodies against the amino and carboxy termini of BRCA1. (B) A western blot probed with an antibody against the Flag epitope. Lanes 2A-2D indicate immunoprecipitation of the Flag-epitope tagged PP1α, β or γ or Flag-Laf4. Lanes 2E-2G show co-immunoprecipitation of Flag-PP1α, β or γ with antibodies against BRCA1, and lane 2H shows a lack of coimmunoprecipitation of the negative control Flag-Laf4 by BRCA1.
0007
PP1 has 3 isoforms encoded by different genes that are 97% conserved across their catalytic domains and distinct roles for each isoform have yet to be determined. When we coimmunoprecipitated Flag-epitope tagged PP1α, β or γ with BRCA1, we observed that all 3 isoforms interacted with BRCA1. Additionally, we have identified the functional PP1 interacting domain within BRCA1. This domain is found in other PP1 regulatory proteins, suggesting that BRCA1 may regulate the activity of PP1 and could act as a scaffold protein to promote the dephosphorylation of BRCA1 associated proteins by PP1.
0104
Consistent with this, our analytical ultracentrifugation (AUC) data showed that BAR-PH protein has a higher dimerization affinity in solution (kd=0.34 nM) than BAR domain alone (kd=0.13 μM; Supplementary data).
0096
To study APPL1−Rab5 interaction in solution, we performed glutathione S-transferase (GST)-mediated pull-down assays. The APPL1 BAR-PH domain (residues 5−385) and a longer fragment with a 40-residue extension downstream of the PH domain, APPL1 (5−419), were each effectively pulled down by GTP-bound GST−Rab5 fusion protein (Figure 4). The APPL1 protein was pulled down by either WT Rab5 preloaded with non-hydrolysable GTP analog (GppNHp) or Rab5-Q79L defective in GTP hydrolysis (with or without preloaded GTP analog), but could not be effectively pulled down by either the WT Rab5 preloaded with GDP or Rab5-S34N defective in GTP binding (Figure 4 and data not shown).
0419
Interestingly, APPL1 would bind to Rab21 in a GTP-dependent manner (Figure 4), indicating that APPL1 is an effector for both Rab5 and Rab21.
0096
To further define the Rab5−APPL1 binding mode, we performed extensive pull-down analyses between variants of Rab5 and APPL1, looking for reversal mutants that could rescue the lost binding ability of others. We identified one such pair; APPL1-N308D abolished the binding to Rab5, while Rab5-L38R had no effect on APPL1 binding. However, Rab5-L38R was found to bind with APPL1-N308D, but not with the other tested APPL1 variants of similar hydrophobic-to-charged mutations, including V25D, A318D, and L321D (Supplementary Figure 6). This result suggests that Rab5-L38R restores binding for APPL1-N308D through complementary, electrostatic, yet specific interactions. It further implies that the position 308 in the β3 strand of APPL1 PH domain is in the vicinity of position 38 in the α1 helix of Rab5 in their complex.
0096
Combined results from our mutagenesis pull-down experiments (Figures 4, 5 and 6), crystal structures of the BAR-PH domain of APPL1 (Figure 1), and structures of GTPase domain of human Rab5 in different nucleotide binding modes (Zhu et al, 2003, 2004) clearly explain the requirement of GTP-bound Rab5 for APPL1 binding. Based on available information, we have modeled the interaction between the two proteins. With the assumption that both proteins remain rigid bodies, our complex model satisfies constraints imposed by the mutagenesis pull-down results (Figure 7).
0419
GTPase binding has emerged as a major function of PH domains in addition to lipid binding (Lemmon, 2004). For example, PH domains in some guanine nucleotide-exchange factors (GEF) have been shown to bind directly to their cognate small GTPases (Rossman et al, 2002, 2003; Lu et al, 2004), and our data now show direct interaction between the APPL1 PH domain and Rab5. So far, only two crystal structures of small GTPase−PH domain complexes are available. One is Ran−RanBD1 (PDB file 1RRP). The interactions between the Ran GTPase domain and RanBD1 PH core domain is fairly minor, occurring between the switch I region of the GTPase (equivalent to the 40's in Rab5) and strand β2 of the PH domain. This interaction alone is unlikely to be sufficient to form a stable complex. Indeed, Ran has a long C-terminal peptide beside the GTPase domain, while the PH domain of RanBD1 has an extra N-terminal peptide. These two terminal peptides wrap around the partner proteins forming the major interaction between Ran and RanBD1. Such an interaction seems not to be required for Rab5 and APPL1, because the GTPase domain of Rab5 and BAR-PH domain of APPL1 are sufficient to mediate their interaction. The second published small GTPase−PH complex is that of Ral−Exo84 (PDB file 1ZC3). In this complex, the PH domain of Exo84 uses L1, β5, and L6 to interact with the interswitch and switch II regions of Ral forming an intermolecular β-sheet extension mediated by the PH β5 strand and GTPase β2 strand (Jin et al, 2005). Our mutagenesis analysis points to a different surface region (β3, L3, and β4) of the PH domain for Rab5 binding. Therefore, the Rab5−APPL1 interaction represents a new GTPase−PH binding mode.
0096
In the Rab5−APPL1 pull-down experiment, 30 μg GST−Rab fusion protein (52 kDa) was incubated with 60 μl of 30% slurry of GSH–Sepharose 4B (GE Healthcare) at 22°C for 30 min. Nucleotide loading reaction was performed on the GSH beads in an exchange buffer of (1 × PBS, 2 mM DTT, 1 mM MgCl2, 4 mM EDTA, and 400 μM GppNHp or GDP) at 22°C for 30 min. Increasing the magnesium ion concentration to 20 mM terminated the loading reaction.
0663
(C) Mutational effects on APPL1 targeting to Rab5-positive early endosomes in the cell. RFP−Rab5-Q79L was coexpressed with GFP−APPL1 (full length, FL; BAR-PH domain; or BAR-PH mutant) in PC12 cells as indicated, followed by confocal fluorescence microscopy. Shown are typical confocal microscopic images indicating the RFP−Rab5-Q79L labeled early endosomes (red) and the colocalization of GFP−APPL1 or mutants (green) in the same cells. Scale bar, 16 μm.
0096
Consistent with prior reports [23,24], we failed to observe stable direct interaction between purified ST and PP2A C subunit using a glutathione S-transferase (GST) pull-down assay (unpublished data).
0004
Soluble fractions were filtered with 0.8 μm syringe filters and applied into a Ni-NTA affinity column pre-equilibrated with 30 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM β-mercaptoethanol. Target protein complexes (the Aα subunit with GST-tag and SV40 ST with His-tag) were eluted with elution buffer (30 mM Tris-HCl [pH 8.0], 50 mM NaCl, 300 mM imidazole, 5 mM β-mercaptoethanol) and dialyzed overnight at 4 °C in 30 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM DTT. Dialyzed protein was applied to a GST affinity column to remove free SV40 ST, and on-column cleavage with TEV protease was performed at 4 °C overnight. The flow-through fraction of the GST column was reapplied into the Ni-NTA column to remove cleaved His-tag and TEV protease.
0416
(B) Subcellular localization of endogenous and exogenous UXT. 293T cells were transfected with (top) or without (bottom) FLAG-UXT. Immunofluorescentmicroscopy was performed with the indicated primary antibodies.
0006
UXT was previously reported to be expressed almost exclusively inside the nucleus of most cells (Markus et al., 2002). This was confirmed in our investigation for either endogenous or overexpressed UXT (Fig. 1 B). To further substantiate its interaction with p65, an in vitro coimmunoprecipitation assay was applied in which full-length HA-p65 and FLAG-UXT proteins were generated and labeled, respectively, with [35S]methionine by in vitro translation. The products were mixed and immunoprecipitated with either control IgG or anti-HA antibody. As shown in Fig. 1 C, UXT could be coprecipitated by antibody against the HA epitope but not by control IgG, which suggests that UXT indeed interacts directly with full-length p65.
0006
To address the physiological relevance of this interaction in mammalian cells, we expressed HA-UXT in 293T cells and then stimulated cells with or without TNF-α for the indicated times. The fractionated cytoplasmic or nuclear extracts were immunoprecipitated with either anti-p65 antibody or IgG as a control, respectively. There was no detectable UXT that interacted with cytoplasmic p65 in the presence or absence of TNF-α (Fig. 1 D), which was consistent with the unique subcellular location of UXT. In addition, there was only a marginal amount of endogenous p65 in the nucleus devoid of TNF-α treatment. Consequently, no UXT was coimmunoprecipitated from this nuclear extract even though there existed a large amount of UXT. In contrast, there exhibited a strong interaction between nuclear p65 and UXT upon TNF-α stimulation. Furthermore, we tested whether endogenous UXT and p65 could interact in response to TNF-α. As shown in Fig. 1 E, endogenous UXT was coimmunoprecipitated by p65 antibody from cells treated with TNF-α. In contrast, UXT was barely detected in the immunoprecipitates without TNF-α treatment. One possible explanation for this phenomenon is that only after p65 translocation into the nucleus could UXT have access to p65. However, we could not formally rule out the possibility that posttranslational modifications of either protein were prerequisites for this interaction in vivo. Collectively, these results indicate that UXT interacts in vivo with p65 upon TNF-α stimulation.
0402
The ChIP assays were performed in terms of A20, IκBα, or GAPDH promoters using antibodies and corresponding primers as described.
0416
Alternatively, we performed immunofluorescence analysis to support this speculation. Interestingly, when transfecting cells with siRNA against UXT and stimulating them with TNF-α, there were an apparently decreased percentage of cells that displayed focused nuclear p65. Approximately 90% of control cells displayed p65 inside the nucleus upon stimulation, whereas only 35–50% displayed p65 in the case of the UXT knockdown specimen (Fig. 5 E).
0402
Given that UXT interacted with p65 and was essential to maintain the presence of NF-κB inside the nucleus, we wondered whether UXT was an integral component of the NF-κB transcriptional enhanceosome in vivo. To address this possibility, we transfected HA-UXT into 293T cells and performed systematic ChIP assays on the promoters of A20 and IκBα as described in Materials and methods. It turned out that UXT was indeed present within the NF-κB transcriptional enhanceosome. Notably, its presence became much more prominent upon stimulation, which suggested that UXT was dynamically recruited onto the enhanceosome. In addition, UXT had nothing to do with the transcription complex on the GAPDH promoter, indicating the selectivity of UXT action (Fig. 7 A). We also stimulated 293T cells and performed similar ChIP assays to confirm again that endogenous UXT was recruited onto the NF-κB enhanceosome in response to stimulation (Fig. 7 B).
0402
After 10 ng/ml TNF-α stimulation, ChIP assays were performed on A20, IκBα, or GAPDH promoters as described.
0412
(C) 293T cells were induced by 10 ng/ml TNF-α for 30 min, and nuclear extracts were prepared and incubated with the indicated antibodies to perform EMSA supershift assays.
0889
For example, p300/CBP played a major role in the acetylation of p65 in vivo (Chen et al., 2002). Conversely, acetylated p65 was subjected to deacetylation by HDAC3 (Chen et al., 2001). Recent work also revealed that SIRT1 physically interacted with p65 and promoted p65 deacetylation (Yeung et al., 2004).
0019
In vitro precipitation experiments demonstrated that COΔB-box was co-immunoprecipitated with GAD:COP1, whereas COΔCCT was not. Therefore, the N-terminal region containing the B-boxes is not required for interaction with COP1, suggesting that the interaction with COP1 is mediated by the C-terminal region of CO that contains the CCT domain.
0402
Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction.
0402
To address this question, we used chromatin immunoprecipitation (ChIP) and amplified the enhanceosome-binding region of the IFN-β promoter by quantitative PCR (Figure 5A). IRF3, absent under non-stimulated conditions, was recruited to the IFN promoter upon infection with L. monocytogenes (Figure 5B). The specificity of this signal was ascertained using a control serum for ChIP.
0676
GS-TAP-tagged DDX3X, isolated by affinity purification using rabbit IgG agarose, was also recruited to the enhanceosome region upon L. monocytogenes infection (Figure 5C). This recruitment was specific because a control region (Figure 5A) could not be amplified by PCR under these conditions. This suggests that DDX3X exerts a direct effect on the IFN-β promoter.
0402
ChIP suggests that DDX3X can be recruited to the IFN promoter, positioning DDX3X downstream of TBK1 at the level of IRF3.
0416
Consistent with these data, S329/T331-phosphorylated MDC1 was detected by immunofluorescence in both control and γ-irradiated cells (Fig 2B; note the absence of staining in cells treated with MDC1 small interfering RNA (siRNA)), confirming that SDTD-phosphorylated MDC1 is present in the absence of damage and also forms IRIF.
0096
Next, we used the recombinant, purified MRN complex in peptide pull-down experiments. These showed that MRN bound to the phosphorylated but not the unphosphorylated form of the SDTD peptide—confirming the direct nature of the interaction—yet did not bind to either version of the H2AX C-terminal peptide (Fig 3A). This indicates the specificity of MRN for the phosphorylated MDC1 SDTD motif and also shows that if, as previously reported, NBS1 binds to γH2AX directly (Kobayashi et al, 2002), this interaction must be less stable than MRN binding to the phosphorylated SDTD motif.
0096
(B) Silver-stained SDS–polyacrylamide gel of an SDTD peptide pull-down. PP, S329 T331 doubly phosphorylated peptide and −, its non-phosphorylated equivalent; B, bead-interacting proteins removed from extracts in a pre-clearing step; M, molecular weight markers.
0096
(B) A 200 ng portion of CK2-phosphorylated or mock-phosphorylated GST-SDTD6 was incubated in pull-down reactions with 25 μl of in vitro-translated (IVT) HA fusions corresponding to amino-acid residues 1–348 of NBS1 (HA-fNBS1), or analogous proteins bearing point mutations predicted to abolish either FHA (R28A/H45A) or BRCT2 (K160M) phosphorylation-dependent interactions.
0096
For the glutathione S-transferase (GST) pull-down experiments, plasmids pGEX-RNP-K (kindly provided by Dr. Levens) and pGEX-4T (GE Healthcare) were used to express GST-hnRNP-K fusion protein or GST alone in Escherichia coli.
0018
pGBT9-p30 and unrelated control protein pGBT9-p54 were independently used as baits to screen a pACT2 cDNA library from pig macrophages in Saccharomyces cerevisiae reporter strain Y190 as previously published [18,20,21]. Yeast were sequentially transformed with bait plasmid and pACT2 library by the lithium acetate method. After auxotrophic and colony size selection, resulting clones were analyzed for expression of GAL4-dependent β-galactosidase. Plasmid DNA from those clones exhibiting β-galactosidase activity was isolated and retransformed into yeast strain Y190 with pGBT9-p30 to eliminate false positives. The sequence of inserts was determined by sequencing using specific primers and compared with the data base of the NCBI using the BLAST program. pGBT9-p30, pGBT9-p54 and pACT2-K were individually transformed in yeast and tested for β-galactosidase activity to exclude activation of gene reporter by itselves.
0096
The interaction was further confirmed by in vitro binding assays using a GST-hnRNP-K fusion protein bound to glutathione-sepharose 4B beads. GST pull-down experiments were carried out followed by Western blot with specific antibodies. First, p30 and another unrelated ASFV protein (in this case, p54 as negative control) were used to bind GST fusion protein or GST alone. Protein p30, and not p54, was retained in the presence of GST-hnRNP K, showing a band of appropriate size (30 kDa) for p30 in Western blotting. This band did not appear in the presence of GST alone, indicating specific interaction of p30 with hnRNP K and not with GST (Fig. 1A). Identical results were obtained in subsequent experiments using BA71V infected or mock-infected cells extracts instead of baculovirus infected cell extracts in the pull-down assay (Fig. 1B).
0096
This interaction was further confirmed by an in vitro GST-fusion pull-down assay, using either p30 obtained from baculovirus system or ASFV infected cell extracts.
0416
Colocalization of p30 and hnRNP-K in the nucleus of infected cells. Cells were infected with BA71V strain and analyzed at different times post infection by confocal immunofluorescence microscopy acquiring 0.1 μm optical sections from the Z-axis. A representative image of an infected cell at 8 hpi is shown (A). p30 was detected with a monoclonal anti-p30 followed by Alexa 647-conjugated goat anti-mouse antibody (red) and hnRNP-K with an anti-hnRNP-K specific serum followed by Alexa 488-conjugated goat anti-rabbit antibody (green). Discrete spots of colocalization of both proteins (orange) can be discerned in the cell nucleus.
0402
Cells were cultured for 24h and prepared using a ChIP assay kit from Upstate Biotechnology, Inc. (Lake Placid, NY) according to the manufacturer's recommendations and preformed as previously describe 28. Primers for the FOXO3a and SCO2 promoter are shown (Supplemental Methods) and their location is shown in supplemental Fig. S3. IP and transient transfection IP westerns were done as previously described 28. Bands for all IPs were detected using an ECL protocol (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with Fuji Las-3000 intelligent darkbox (FujiFilm Medical Systems, Stamford, CT).
0006
We sought to establish whether the mitochondrial localized SIRT3 may also form a physical interaction with the FOXO family protein, FOXO3a, using co-immunoprecipitation (Co-IP) techniques. Carboxy-terminally myc tagged wild-type (p-myc-hSIRT3-wt), and mutant (p-myc-hSIRT3-mt) SIRT3 expression vectors were transfected into Cos-7 cells followed by Co-IP with an anti-myc antibody.
0402
ChIP analysis showed that wt-SIRT3 cells have an increase in FOXO3a binding to upstream regulatory regions of gene promoters that each contain two canonical FOXO3a binding sites roughly 1 kb upstream of the transcription start site (supplemental Fig. S3). FOXO3a binding to both the MnSOD (Fig.3D, upper panel) and SCO2 (lower panel) promoters is increased in the wt-SIRT3, as compared to mt-SIRT3 cells. These experiments imply that SIRT3 may increase FOXO3a DNA-binding.
0006
(A) FOXO3a binds to SIRT3 in vitro. Cos-7 cells were transfected with either SIRT3 wild-type (p-myc-hSIRT3-wt) or deacetylation mutant (p-myc-hSIRT3-mt) vectors and cell lysates were immunoprecipitated (IPd) with an anti-Myc antibody followed by Western analysis with an anti-FOXO3a antibody. (B) HCT116 cell lysates were IPd with either an anti-FOXO3a or anti-SIRT3 antibody, resolved by SDS-PAGE, and immunoblotted with anti-FOXO3a antibody. (C) Mitochondrial factions from HCT116 cells were IPd with either an anti-FOXO3a or anti-SIRT3 antibody and immunoblotted with anti-FOXO3a antibody.
0402
(D) ChIP analysis of FOXO3a finding to the MnSOD and SCO2 promoters in HCT116 cells that overexpress either a wild-type or deacetylation null SIRT3 gene.
0006
Cells were fixed with 1% formaldehyde to crosslink protein-DNA interactions, sonicated, and fixed cells were immunoprecipitated with either an anti-FOXO3a antibody.
0030
Kelch motifs, structural repeats first observed in the Drosophila actin cross-linking protein kelch, allow protein folding into a cylindrical ‘β-propeller structure’ (Adams et al., 2000) forming a potential protein–protein interaction domain (Andrade et al., 2001).
0007
Co-immunoprecipitation assays were used to confirm the protein–protein interactions. Subsequently, localization of ACBP4 and its interacting protein, AtEBP, was confirmed using transient expression of GFP- and DsRed-tagged fusion proteins in Nicotiana tabacum.
0018
S. cerevisiae strain YPB2 was transformed with bait plasmid pAT188 and transformants were plated on synthetic dextrose agar plates lacking leucine [SD-leu]. An aliquot of transformants was also tested on [SD-leu-his] medium supplemented with 10 mM 3-amino-1, 2, 4-triazole (3-AT) because an absence of growth on this medium would confirm that the DB-‘bait’ fusion protein is unable to initiate transcription of HIS3. Subsequently, the bait-carrying strain was tested negative for β-galactosidase activity using the X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) colony filter assay. This further showed that the bait was not able to activate transcription of the lacZ reporter gene. The prey vector pBI-771, a variant of pPC86 (Chevray and Nathans, 1992; Kohalmi et al., 1998), was introduced into this strain and its inability to grow on [SD-leu-trp-his] medium supplemented with 10 mM 3-AT and its lack of β-galactosidase activity were confirmed before the bait was further used in cDNA library screening.
0663
Tobacco leaf epidermal cells from agroinfiltration were examined under a Zeiss LSM 510 inverted confocal laser-scanning microscope (Zeiss, Jena, Germany) following the settings described by Goodin et al. (2002) with minor modifications. Single optical sections were scanned as resulting images for each transient expression. For each plasmid construct, 10–15 cells were imaged with similar results. GFP fluorescence was excited at 488 nm, filtered through a primary dichroic (UV/488/543), a secondary dichroic of 545 nm, and subsequently through BP505–530 nm emission filters to the photomultiplier tube (PMT) detector. DsRed fluorescence was excited at 543 nm, the emission was passed through similar primary and secondary dichroic mirrors and finally through a BP560–615 nm emission filter to the PMT detector. Fluorescence resonance energy transfer (FRET) pairs GFP/DsRed were analysed using a confocal laser-scanning microscope (Zeiss LSM510 META). FRET measurements of DsRed emission with zero contribution from GFP, was accomplished as described by Erickson et al. (2003) using the following settings: excitation at 488 nm and emission filters, BP 505–530 nm for GFP and BP 600–637 nm for DsRed.
0040
Arabidopsis leaves were fixed in a solution of 4% (v/v) paraformaldehyde and 0.5% (v/v) glutaradehyde in 0.1 M phosphate buffer (pH 7.2) for 20 min under vacuum and then a further 3 h at room temperature. The specimens were then dehydrated in a graded ethanol series, infiltrated in stepwise increments of LR white resin (London Resin, Theale, Berkshire, UK) and polymerized at 45 °C for 24 h. Materials for immuno-gold labelling were prepared according to the procedure of Varagona and Raikhel (1994) with the modification as described. Specimens (90 nm) were sectioned using a Leica Reichert Ultracut S microtome and mounted on formvar-coated slotted grids. Grids were incubated in a blocking solution of TTBS containing 1% (w/v) fish skin gelatin and 1% (w/v) BSA for 30 min. Anti-ACBP4 antibodies diluted 1:50 in blocking solution were added and incubated at room temperature for 2 h. The grids were then rinsed three times, each for 5 min, in TTBS and then incubated with 10 nm gold-conjugated goat anti-rabbit IgG secondary antibody (Sigma), diluted 1:20 with blocking solution. Grids were rinsed three times, each for 5 min in TTBS, following by three 5-min rinses in distilled water. After being stained in 2% (w/v) uranyl acetate for 6 min followed by 2% (w/v) lead citrate for 6 min, the sections were visualized and photographed using Philips EM208s electron microscope operating at 80 kV. Controls were performed excluding the primary antibody.
0040
(B, C, D) Immuno-gold labelling of ACBP4 in an Arabidopsis leaf cell using transmission electron microscopy. Transverse sections were stained with affinity-purified ACBP4-specific antibodies. (B) Transverse sections of leaves stained with ACBP4-specific antibodies. (C) Magnification of the boxed area in (B). (D) Control labelling of a leaf cell using secondary antibodies alone. Arrowheads, gold particles. V, vacuole; C, cytosol; Ch, chloroplast; N, nucleus; Cw, cell wall; Bars in (B) represent 2 μm, and in (C, D), 0.2 μm.
0040
Immuno-electron microscopy was carried out using transverse sections of leaves of 2-week-old Arabidopsis germinated and grown in MS medium under a 16/8 h light/dark regime. Although immuno-gold labelling with the anti-ACBP4 antibodies was mostly evident in the cytosol, some signals were detected at the periphery of the nucleus, (Fig. 3B, C). In the control, when the primary antibody was replaced by blocking solution, no significant immuno-gold labelling was observed (Fig. 3D). The immunolocalization of signals at the periphery of the nucleus may have culminated from the interaction of ACBP4 with AtEBP.
0040
In this study, GFP:AtEBP was not confined to the nucleus but was also detected in the cytosol where it could interact with ACBP4. ACBP4:DsRed, transiently-expressed in tobacco leaves, was predominantly targeted to the cytosol but immuno-electron microscopy indicated localization of ACBP4 in the cytosol with signals detected at the periphery of the nucleus, perhaps as a consequence of its interaction with AtEBP.
