Figure 4.

Bak displaced from Mcl-1 associates with pBcl-2 in resistant cell lines. (A) K562, HL60 and U937 cells were lysed. Mcl-1, Bcl-2 and pBcl-2 immunoprecipitations were performed, and immunoprecipitated fractions were analysed by western blotting for the indicated proteins. (B) Isothermal calorimetric titration of the Bcl-2 phosphorylation mutant protein, EEE-Bcl-2, with the Bak protein. The EEE-Bcl-2 concentration was 30 μℳ, and the Bak concentration was 300 μℳ. Injection volumes were 20 μl. (C) Immunoprecipitation of K562 cell lysates. Cells were treated in the presence or absence of 5 μℳ S1 for 8, 18 and 24 h. Bim, Bax, Bak, Bcl-2 and Mcl-1 levels were analysed by western blotting. (D) Immunoprecipitation of U937 cell lysates using the indicated antibodies. (Left panel) The immunoprecipitates were analysed for the presence of the indicated proteins by immunoblotting. Cells were treated in the presence or absence of 20 μℳ S1 for 8, 18 and 24 h. (Middle panel) U937 cells were treated in the presence or absence of 20 μℳ S1 for 24 h. Supernatants of the Mcl-1 immunoprecipitation were used for the pBcl-2 immunoprecipitation. Immunoblotting was performed as described previously. (Right panel) U937 cell lysates were detected by immunoblotting using the indicated antibodies. Ab, antibody.