FIGURE 2.

MPK38/ASK1 and PDK1 signaling decreases MPK38-PDK1 binding. HEK293 cells were incubated with or without wortmannin (100 nm, 30 min) and then treated with insulin (100 nm, 20 min). The cell lysates were then immunoprecipitated (IP) with either rabbit preimmune serum (Preimm.) or an anti-MPK38 antibody (α-MPK38) followed by immunoblotting with an anti-PDK1 antibody to determine endogenous MPK38-PDK1 binding (1st panel). Equal amounts of cell lysate were treated with or without the following stimuli (20): H₂O₂ (2 mm, 30 min), TNF-α (500 ng/ml, 30 min), thapsigargin (Tg) (20 μm, 30 min), or ionomycin (IONO) (1 μm, 24 h). The cell lysates were then immunoprecipitated with the indicated rabbit preimmune serum or anti-MPK38 antibody (α-MPK38) and immunoblotted with an anti-PDK1 antibody to assess endogenous MPK38-PDK1 binding (2nd to 4th panels). WB, Western blot.