FIGURE 4.
Participation of Bad in the induction of adhesion-dependent apoptosis. A, dephosphorylation of Bad through TNIIIA2-induced adhesion of Ramos cells to FN. Ramos cell suspension (3 × 106 cells) with or without TNIIIA2 (25 or 50 μg/ml) was seeded on a plate coated with FN and then incubated in the presence or absence of CS-1 (CS) (200 μg/ml). After incubation for 16 h, cell lysates with equal amounts of protein were subjected to Western blot analysis using anti-phospho-Bad or anti-Bad, as described under “Experimental Procedures.” B, dissociation of Bad from binding with Bcl-xL through TNIIIA2-induced adhesion of Ramos cells to the FN. The cells were cultured on an FN-coated plate in the presence or absence of TNIIIA2 (50 μg/ml) for 16 h. The cell lysates were immunoprecipitated with anti-Bcl-xL Ab, and the resulting precipitate was subjected to Western blot analysis with anti-Bad Ab or anti-Bcl-xL, as described under “Experimental Procedures.” C and D, effect of knockdown of Bad protein expression by siRNA on TNIIIA2-induced apoptosis. Transfection of Bad siRNA (Bad) or control siRNA (Cont) was performed as described under “Experimental Procedures.” Cell proliferation/survival assay was performed as in Fig. 2. Two representative data (Experiments 1 and 2) of four independent experiments are shown. *, p < 0.005 compared with control. IP, immunoprecipitation; IB, immunoblot.