Fig. 6. SSRP1 co-resides with p63γ at the p53RE sites of the endogenous MDM2 and p21waf1 promoters. (A) ChIP analyses. H1299 cells were transfected with plasmids encoding no protein (3 µg), or with Myc-p63γ (1 µg) and/or Flag-SSRP1 (2 µg), as indicated on top. At 36 h post-transfection, cells were treated with a 1% formaldehyde solution for ChIP assays as described in Materials and methods. PCR products of 117 bp (top) encompassing the p53RE motif of the MDM2 promoter, 105 bp (middle) of the p21waf1/cip1 promoter or 98 bp (bottom) of the non-p53RE sequence 2.5 kb upstream from the p53RE site of the MDM2 promoter were tested from the immunoprecipitates using antibodies against either Myc or Flag, as indicated on top. (B) Western blot analysis of the same transfected cells. Cell lysates containing 150 µg of proteins were loaded onto an SDS gel. Myc-p63γ and Flag-SSRP1 were detected with antibodies against Myc and Flag. (C) SSRP1 directly binds to the p53RE-bound p63γ. EMSAs were carried out as described in Materials and methods. In the reactions in lanes 1–9, 300 ng of poly(dIdC), 100 ng of wild-type (lane 2) or mutant (lane 3) p53RE oligomers, 500 ng of p63γ, 200 and 400 ng of SSRP1 (lanes 4 and 5), and 200 and 400 ng of BSA (lanes 6 and 7) were used as indicated on top. In the reactions in lanes 10–15, 400 ng of SSRP1 and 500 ng of anti-SSRP1 or anti-HA peptide antibodies were used in the presence or absence of 100 or 200 ng of poly(dIdC) as indicated on top.