Figure 1.

Hyperglycemia increases p62 and PKCζ activation in VSMCs. VSMCs were cultured in DMEM containing normal glucose (5 mM) plus 10% FBS then serum deprived for 16 h before exposure to 25 mM glucose for the indicated times. A) Cell lysates were immunoblotted (IB) with an anti-p62 antibody. To control for loading, the blot was stripped and reprobed with an anti–β-actin antibody. B) Cell lysates were immunoblotted with an anti-pThr410 PKCζ antibody. The blot was stripped and reprobed with an anti-PKCζ antibody. The value of each bar is the ratio of the scan values of the p62 or pThr410 PKCζ bands divided by the values of β-actin or the PKCζ bands. C) PKCζ was immunoprecipitated with an anti-PKCζ antibody, and the immune complexes were used to measure in vitro PKCζ kinase activity following the procedure described in Materials and Methods. To control for PKCζ input, after immunoprecipitation, the amount of immune complex from each treatment was adjusted for PKCζ as determined by immunoblotting with anti-PKCζ. High glucose (HG) was 25 mM, and normal glucose (NG) was 5 mM. *P < 0.05 and **P < 0.01 indicate significant differences between 2 treatments.