Figure 4.

PDK1 phosphorylates PKCζ contained in the p62 complex during hyperglycemia. VSMCs were cultured in DMEM containing NG plus 10% FBS then serum deprived for 16 h before exposure to 25 mM glucose (NHG) for 6 h or maintained in NG. A and B) Cell lysates were immunoprecipitated (IP) with an anti-p62 antibody and immunoblotted (IB) with an anti-PDK1 or PDK1 (Ser241) antibody. To control for loading, the same amount of cell lysate was immunoblotted with an anti-PDK1 or anti-p62 antibody. C) Different concentrations of GSK227434 were added 30 min before a 6 h exposure to SFM containing 25 mM glucose. Cell lysates were immunoblotted with anti-pThr410 PKCζ and PDK1 (Ser241) antibodies. The blots were stripped and reprobed with an anti-PKCζ or anti-PDK1 antibody. D) VSMCs expressing the shRNA targeting LacZ (Ctrl Si) and p62 (p62 Si) were cultured in DMEM containing normal glucose (5 mM, NG) plus 10% FBS then serum deprived for 16 h before exposure to 25 mM glucose (NHG) for 6 h or maintained in NG. Cell lysates were immunoprecipitated with an anti-PKCζ antibody and immunoblotted with an anti-PDK1 antibody. To control for loading, the blots were stripped and reprobed with an anti-PKCζ antibody. E) Cell lysates were immunoprecipitated with an anti-PKCζ antibody and immunoblotted with an anti-PDK1 antibody. To control for loading, the blots were reprobed with an anti-PKCζ antibody.