Figure 3.

Knockdown or deletion of p62 prevents hyperglycemia-stimulated PKCζ activation. A) VSMCs expressing the shRNA targeting LacZ (Ctrl Si) and p62 (p62 Si) were cultured in DMEM containing 25 mM glucose. Cell lysates were immunoblotted (IB) with an anti-p62 antibody. To control for loading, the blots were stripped and reprobed with anti–β-actin. B and C) VSMCs expressing Ctrl Si or 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 immunoblotted with an anti-p62 antibody and reprobed with an anti–β-actin antibody (B) or immunoblotted with anti-pThr410 PKCζ, anti-PKCζ, or anti-PDK1 antibodies (C). D) VSMCs were cultured as in (B) and (C). Cell lysates were immunoprecipitated (IP) with an anti-p62 antibody. The immune complexes (Pellet) and supernatant (Super.) were immunoblotted with anti-pThr410 PKCζ and anti-PKCζ antibodies.