Protein was immunoprecipitated using antibodies against BRCA1, or an antibody against the Flag epitope to immunoprecipitate Flag epitope tagged PP1α, β, or γ. BRCA1 coimmunoprecipitated all three PP1 isoforms, and conversely, PP1 α, β and γ coimmunoprecipitated BRCA1 (Figure 2), indicating that the interaction between BRCA1 and PP1 is specific.(D) 293T cells were transfected with HA-UXT. 24 h after transfection, cells were treated with 10 ng/ml TNF-α for the indicated times and fractionated to cytoplasmic and nuclear fractions, which were immunoprecipitated and immunoblotted with the indicated antibodies, respectively. (E) 293T cells were treated with 10 ng/ml TNF-α for the indicated times. Whole cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Bar, 10 μm.
To explore the UXT-binding region within p65, we constructed a series of p65 deletion mutants (Fig. 2 A). It was found that the loss of amino acids 1–285 at the N terminus of p65 resulted in its complete inability to interact with UXT (Fig. 2 B, top). In contrast, p65 fragments spanning amino acids 1–286, 1–312, or 1–372 fully retained their binding capability and interacted with UXT as well as the wild type (Fig. 2 B, middle). Because the RHD of p65 consisted of two Ig-like domains (Chen et al., 1998), we made two additional deletion mutants of p65 (amino acids 1–190 and 191–551), each of which contained only one Ig-like domain. Immunoprecipitation assays revealed that neither of them was able to interact with UXT (Fig. 2 B, bottom).
(B) Tagged full-length UXT was transfected into 293T cells along with p65 and its deletion mutants as indicated. Whole cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (C) 293T cells were transfected with FLAG-UXT together with HA-p50, myc-cRel, and HA–lymphoid enhancer binding factor 1. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies.Therefore, whether COP1 and CO interact in vitro was tested using a co-immunoprecipitation assay (Figure 3). COP1 attached to the GAL4 activation domain (GAD:COP1) and CO were made in an in vitro transcription/translation system and combined. GAD:COP1 was precipitated with anti-GAD antibody and CO was co-precipitated with GAD:COP1 (Figure 3).
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.
COP1 directly interacts with target proteins and directs them for degradation (Hoecker, 2005; Jiao et al, 2007). CO is composed of three domains, zinc-finger B-boxes, a central domain and the C-terminal CCT domain (Wenkel et al, 2006). CO and COP1 interact directly in vitro as demonstrated by immunoprecipitation experiments. This interaction was almost abolished when the C-terminal part of CO was removed, suggesting that COP1 interacts with the C-terminal region of CO, as was previously observed for interactions between COP1 and COL3 or between CO and SPA1 (Datta et al, 2006; Laubinger et al, 2006).
In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments.Immunoprecipitation experiments using tagged TBK1 suggested that its interaction with DDX3X and the transcription factor IRF3 are significantly weaker than the interaction between TBK1 and TANK and therefore not detected by coimmunoprecipitation under stringent conditions (Supplementary Figure 1A).The wild-type MDC1 derivative efficiently co-immunoprecipitated MRN at physiological salt concentrations, whereas only low levels of MRN were recovered in immunoprecipitates of the MDC1SDTDΔ mutant (Fig 4B), consistent with the SDTD region being the principal MRN interaction interface.
(B) Indicated expression constructs were transfected into human embryonic kidney 293 cells. After 48 h, extracts were prepared, immunoprecipitated (IP) with GFP antibodies and immunoblotted. Immunoprecipitations and washes were performed at 150 mM salt; INP, input (5%). (C,D) Indicated osteosarcoma (U2OS) cell lines were treated with two rounds of control (CNTL) or MDC1-targeting siRNA for 72 h. Cells were then treated with 5 Gy of X-rays and processed for immunofluorescence 4 h later with MDC1, (C) NBS1 or (D) 53BP1 antibodies (non-irradiated cells are shown in supplementary Fig S4B,C online).Co-IP experiments were repeated using the HCT116 SIRT3 expressing stable cell lines. FOXO3a was found to interact with SIRT3 in both whole cell (Fig. 1B) and mitochondrial extracts (Fig. 1C). All fractions and samples in the Co-IP experiments were checked for the presence of SIRT3, using a SIRT3 specific antibody (Biomol, Plymouth Meeting , PA), as well as tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), and Cytochrome C (Mitosciences, Eugene, OR) to ensure fraction purity (data not shown). These experiments demonstrate that both wild type and mutant SIRT3 form a physical interaction with FOXO3a in mitochondrial extracts.To corroborate the interaction from yeast two-hybrid analysis, co-immunoprecipitation studies were performed according to Mongiat et al. (2003). All constructs used in these interaction assays were derivatives of vector pBluescriptII KS(–) (pKS). The HindIII-SacI fragment from pBI-771 carrying GAL4(TA) (amino acids 768–881) was cloned into corresponding restriction sites on pKS. The GAL4(TA)-ACBP4 fusion construct was prepared by inserting ACBP4 cDNA from pAT181, on a 2 kb EcoRI-BamHI fragment, into the EcoRI-BglII sites of pKS-TA with the 5′ of TA-ACBP4 adjacent to the T3 promoter.
Co-immunoprecipitation with monoclonal anti-GAL4(TA) antibody (Clontech, USA) was performed following Mongiat et al. (2003).
(B) Co-immunoprecipitation of ACBP4 and AtEBP using the anti-GAL4(TA) monoclonal antibody. Autoradiograph of a 12% SDS-PAGE (left panel) showing the in vitro transcribed and translated ADF3, AtEBP, and GAL4(TA)-ACBP4, respectively, as indicated. The right panel shows the co-immunoprecipitation of equimolar amounts of GAL4(TA)-ACBP4 and ADF3 or AtEBP using the anti-GAL4(TA) antibody. Arrows indicate the positions of these proteins.
Co-immunoprecipitation of in vitro transcription/translation products to the GAL4(TA)-ACBP4 fusion protein, immobilized to protein A/agarose beads, using monoclonal antibody against GAL4(TA), showed that the GAL4(TA)-ACBP4 fusion protein significantly binds AtEBP (Fig. 1B). However, no binding of GAL4(TA)-ACBP4 to ADF3 was observed (Fig. 1B), perhaps due to the lack of cofactors which must be present for their in vitro interaction.
The interaction of AtEBP and ACBP4 was further substantiated by co-immunoprecipitation and by using autofluorescent protein fusions in the transient expression of tobacco leaf epidermal cells. ACBP4 and AtEBP showed overlapping expression patterns in leaves and stems and both were inducible by ACC, MeJA treatment, and infection with the fungal pathogen, Botrytis cinerea.