Figure 3

Nuclear PKM2-induced STAT3 phosphorylation correlates with gefitinib resistance in CRC cells.

(A) Representative CRC cell lines were treated with gefitnib or left untreated. At 48 h after treatment, the cells were lysed and subjected to immunoblot analysis. The levels of phosphorylated and total STAT3 in nuclear extracts (NE) and whole-cell lysates (WCL) were detected. (B) Total and phosphorylated STAT3 were detected with Western blotting in HT29 or C2BBel cells, which were transfected with PKM2-NLS or PKM2 shRNA, respectively. (C) STAT3 phosphorylation was regulated by nuclear PKM2 through physical binding. The left panel shows Western blotting results of STAT3 after PKM2 immunoprecipitation (IP); the middle and right panel shows Western blot results for the reciprocal IP and loading control. (D) Phosphorylation of endogenous STAT3 (IP: STAT3) or exogenously expressed mutant Y705A (IP: Flag) by PKM2 in SW480 cell nucleus was analyzed by immunoprecipitation using anti-STAT3 antibody (IP: STAT3) or anti-Flag antibody (IP: Flag) followed by immunoblot using antibody against phospho-tyrosine (IB: pTy-100).