Figure 3
Co-immunoprecipitation assay. TBP/HAP1-B155–582 interactions in co-transfected cells. (A) 293 cells were co-transfected with pCMV-HA-TBP-FL and pCMV-MYC-HAP1. Whole cell lysates (WCL) (lanes 1–2, where lane 1 and lane 2 represent 4% of each WCL used for immunoprecipitation in lanes 3 and 4, respectively) or immunoprecipitated samples (lanes 3–4) were assayed using western blots with anti-MYC antibody. MYC-tagged HAP1 co-precipitated with TBP in the presence of the anti-TBP antibody (lane 3), but not with the non-specific antibody (lane 4). (B) COS-7 cells were co-transfected with pFLAG-CMV-GFP-TBP-FL (lanes 1–4, 7–8) or pFLAG-CMV-GFP (lanes 5–6, 9) and pCMV-MYC-HAP1-B155–582 (lanes 1–9). Whole cell lysates (lanes 1, 3, and 5, HAP1) or immunoprecipitated samples (lanes 2, 4, and 6, HAP1) were assayed using western blots and anti-MYC antibody. MYC-tagged HAP1 co-precipitated with FLAG-tagged-GFP-TBP in the presence of the anti-FLAG antibody (lane 2), but not with the non-specific antibody (lane 4). Myc-tagged HAP1 did not co-precipitate with FLAG-tagged-GFP in the presence of anti-FLAG antibody (lane 6). 30% of each immunoprecipitated sample was also blotted with anti-FLAG antibody (lanes 7–9) to verify the amount of FLAG-tagged protein that was captured in each co-precipitation assay (lanes 7–9 represent co-precipitated samples in lanes 2, 4, and 6, respectively).