FIGURE 3.
Effects of Cholix on Bcl-2 family proteins. A, Cholix-induced conformational changes and association of Bak and Bax in HeLa cells. Cells (2 × 105 cells/well) in a 6-well dish were grown for 24 h and then incubated with wild-type (WT) or mutant (MT) Cholix (10 μg/ml) for the indicated duration times. Cells were lysed and immunoprecipitated with anti-conformation-specific Bak (cBak) or Bax (cBax) antibodies as described in the previous report (19). The top panel shows the total cell lysate (TCL), and the lower panel shows Bak and Bax, which were immunoprecipitated with anti-cBak or anti-cBax antibodies, respectively. Data are representative of three separate experiments. B, effects of protein synthesis inhibition induced by Cholix on anti-apoptotic Bcl-2 family proteins. Cells (1 × 105 cells/well) in a 12-well dish were grown for 24 h and then incubated with 10 μg/ml Cholix WT for 0, 3, 6, and 9 h or with MT for 9 h. The expression levels of Bcl-2, Bcl-XL, and Mcl-1 in the total cell lysate were detected by Western blotting. Data are representative of three separate experiments.