TAp63 regulates Bcl-2 expression. (A-B) HEK-293 cells were transfected with empty, p63TAα, and p63TAγ constructs for 24 hours. (A) Total RNA was isolated, and RT-PCR for Bcl-2 was performed as described in “RNA isolation and reverse transciption.” The results presented are representative of 4 different experiments. (B) Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on 10% (wt/vol) SDS-PAGE and blotted with anti–Bcl-2 antibody, followed by HRP-conjugated antimouse antibodies. The membrane was stripped and reblotted with antitubulin. The arrow indicates the Bcl-2 band. The results presented are representative of 3 separate experiments. (C) ChIP analysis of p63 binding to the Bcl-2 promoter. HEK-293 cells were transfected with empty, p63TAα, and p63TAγ constructs for 24 hours. Chromatin prepared from these cells was immunoprecipitated with control and anti-p63 antibodies. Presence of the promoter sequence was then quantified by RT-PCR. (D) Primary B cells were transfected for 8 hours with various constructs of p63 using AMAXA reagents, as described in “Cell transfection.” RNA was isolated and Bcl-2 transcription was determined by RT-PCR. The results presented are representative of 3 different experiments. (E-H) siRNA for p63 or lacZ (control) was transfected into primary B cells in the presence or absence of anti-CD74 (E) or MIF (G,H) stimulation. Total RNA was isolated, and RT-PCR was performed as described in “RNA isolation and reverse transciption.” The results presented are representative of 4 different experiments (E,G). Annexin staining of B220⁺ cells was performed (F). Annexin and PI staining of B220⁺ cells was performed (H). The intensity of the p63 band was calculated as described in Figure 2. Numbers on the plots in panel H are percentages of total cells.