Figure 2.
p73-TAD binds to anti-apoptotic Bcl-XL. a, the interactions of p73-TAD and Bcl-XL, Bcl-2, and Bcl-w as determined by a GST pulldown assay. Lysates from HEK293T cells transfected with GST-tagged p73-TAD, and the indicated FLAG-tagged plasmids were subjected to GST–p73-TAD pulldown followed by immunoblotting with an anti-FLAG antibody. b, the interaction between TAp73 or p73-TAD and Bcl-XL. H1299 cells transfected with the indicated plasmids were immunoprecipitated (IP) with an anti-FLAG antibody and probed with an anti-p73 antibody. c, the interaction between overexpressed TAp73 or p73-TAD and endogenous Bcl-XL. Lysates from GFP-TAp73 overexpressed H1299 cells were immunoprecipitated with an anti–Bcl-XL antibody and probed with an anti-GFP antibody. The same input sample was divided into two fractions, each of which was then used for immunoprecipitation in lanes 1 and 2 (IgG) or lanes 3 and 4 (Bcl-XL). d, the interaction between endogenous TAp73 and Bcl-XL or Bcl-2. H1299 cells were treated with 50 μm etoposide, and then the cell lysates were immunoprecipitated with an anti–Bcl-XL or anti-Bcl-2 antibody and probed with an anti-p73 antibody. e, p73-TAD liberates a pro-apoptotic BH3-only protein Bid from Bcl-XL. Immunoprecipitation experiments were performed to detect the competition of TAp73 with Bid for binding to endogenous Bcl-XL. HEK293T cells were transfected with pCMV–Myc–Bid and peGFP–C3–TAp73. The cell lysates were immunoprecipitated with an anti–Bcl-XL antibody and probed with the indicated antibodies. f, immunoprecipitation experiments were performed to detect the competition of p73-TAD with Bid for binding to endogenous Bcl-XL. HEK293T cells were transfected with pCMV–Myc–Bid and peGFP–C3–p73-TAD.