Fig 2.

Akt/PKB promotes phosphorylation of ASK1 at Ser967. (a to c) COS7 cells were transfected with HA-ASK1, wild-type Akt (Akt WT), constitutively active Akt (Akt ΔPH), and/or catalytically inactive Akt (Akt KM). (a) At 48 h after transfection, the cells were serum starved for 24 h. (b) COS7 cells transfected with HA-ASK1 (WT) or HA-ASK1 S967A (SA) grown in the presence of serum were treated with wortmannin (200 nm), LY294002 (30 μM), Akt inhibitor 124005 (40 μM), or dimethyl sulfoxide (DMSO; vehicle) for 30 min. (c) At 48 h after transfection, the cells were serum starved for 24 h and treated with IGF-1 (100 ng/ml) for 20 min. (d) An in vitro radiolabeling kinase assay for ASK1 phosphorylation was performed with recombinant Akt. The total protein levels were analyzed by Coomassie blue staining. The pS967 ASK1 and total protein levels in panels a to c were analyzed by Western blotting.