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. 2009 Nov 17;285(4):2397–2414. doi: 10.1074/jbc.M109.064295

FIGURE 1.

FIGURE 1.

PDK1 forms a complex with ASK1 in vivo and in vitro. A, HEK293 cells were transiently transfected with the indicated combinations of expression vectors encoding GST-PDK1 and FLAG-ASK1. GST fusion proteins were purified with glutathione-Sepharose beads (GST Purification). Complex formation between PDK1 and ASK1 was determined by immunoblot analysis with an anti-FLAG antibody. B, proteins immunoprecipitated from HEK293 and HeLa cell lysates, with either rabbit preimmune serum (Preimm.) or anti-PDK1 antibody (α-PDK1), were immunoblotted with an anti-ASK1 antibody to determine the association between endogenous PDK1 and ASK1. C, for the in vitro association between PDK1 and ASK1, autophosphorylated recombinant PDK1 (2–3 μg) was incubated with unlabeled recombinant GST or GST-ASK1(K709R) (5 μg each) at room temperature for 1 h and analyzed on an 8% native polyacrylamide gel. WB, Western blot; IP, immunoprecipitation.