FIG. 2.

Akt interacts with ASK1 in cells. (A) Endogenous association of Akt and ASK1 in L929 cells. Cells were serum starved for 36 h and then treated with or without 100 ng of IGF-1/ml for 7 min. Endogenous Akt was immunoprecipitated (IP) from lysates with anti-Akt, and proteins were detected with anti-ASK1 H-300 and anti-Akt. To confirm that both active and inactive Akt were immunoprecipitated, membranes were stripped and reprobed with anti-phospho-Akt(S473) (anti-pAkt). As a positive control, crude lysate (left) was probed for ASK1 and Akt by immunoblotting. Aktp represents immunoprecipitation with anti-Akt antibodies preincubated with an Akt peptide. All data are representative of three independent experiments. W, Western blot. (B) Akt and ASK1 associate independently of ASK1 serine 83 phosphorylation in transfected 293 cells. Cells were transfected with 1 μg each of the indicated constructs for 36 h, and lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (C) Akt and ASK1 binding occurs independently of kinase activity in transfected 293 cells. Samples were processed as for panel B.