FIGURE 4.

Kinase inhibitors indicate direct involvement of RAF kinases in BAD phosphorylation. HEK293 cells were transiently transfected with BAD and B-RAF expression vectors. 16 h post-transfection, cells were cultivated in medium supplemented with 0.3% serum. During that time, cells were additionally treated with various concentrations of MEK inhibitors PD98059, U0126, and CI1040 (A), with PI3K inhibitors wortmannin and LY294002 (B), with suramin (C), and with RAF inhibitor (BAY 43-9006) (D) for 22 h, respectively. Phosphorylation of BAD was detected with phosphospecific BAD antibodies. The efficiency of the PI3K inhibitors has been verified by abolition of Akt-S473 phosphorylation in stimulated HEK293 cells (B). Treatment with the RAF inhibitor BAY 43-9006 prevented both BAD and ERK phosphorylation in a concentration-dependent manner. These experiments were repeated three times with the same results.