Figure 4.

Activation of conventional PKC by high P loading in BAECs. (A) Effect of high P loading on the PKC activity of BAECs. We treated BAECs with control (0.9 mM P), 1.5 mM P, 3 mM P, or 3 mM P with 200 μM PFA for 15 min, then pretreated whole cell lysates, and subjected lysates to measurement of conventional PKC activity using the TruLight PKCα Assay kit. *P < 0.05 versus column 1, ^†P < 0.05 versus 3 mM P (column 3). (B) Effect of high P loading on the subcellular localization of PKCα in BAECs. We treated BAECs with the indicated concentration of P or glucose in Medium 199 medium without serum for 1 h, and then fractionated the cells into cytosol and caveolar membrane fractions. We separated 20 μg of protein of each fraction by SDS-PAGE and performed western blot analysis with anti-PKCα mAb and anti-caveolin pAb. The data are representative from two separate experiments.