(a) Characterization of PKC isoform and activities in PKC-δ isoform–overexpressed cells. (b) Immunoblot analysis of PKC isoforms in PKC-δ−overexpressed and the control cells containing the retroviral vector without PKC-δ cDNA derived from rat aortic SMC. The infection was performed using the retroviral vector pBabe-Puro, as described in Methods. After the vector with or without PKC-δ, cDNA was transfected into BOSC23 cells via Lipofectamine, and the supernatant was filtered and added to primary cultured rat SMC for infection. Positive transfectants were selected using puromysin. PKC proteins in both membranous and cytosolic fractions were partially purified. The proteins were separated by 8% SDS-PAGE, and immunoblot analysis was performed using various types of antibodies on PKC isoforms as described in Methods. The results are derived from four separate experiments. Each bar represents the mean ± SEM. *P < 0.05, **P < 0.01 vs. control cells at 5.5 mM glucose; ^#P < 0.05 vs. PKC-δ−overexpressed cells at 5.5 mM glucose. (b) Effect of PKC-δ overexpression on p38 MAP kinase activity in rat aortic SMC. After 72 h of exposure to 5.5 mM or 16.5 mM glucose, the cells were untreated or treated with a PKC-specific inhibitor, GF109203X (GFX, 5 μM), for 30 min, and then lysed. The p38 MAP kinase activity was quantitated by the phosphorylation of MBP using [γ-³²P]ATP as described in Methods. The results were derived from four separate experiments, with each experiment performed in duplicate. Each bar represents the mean ± SEM. **P < 0.01 vs. control at 5.5 mM glucose; ^#P < 0.05, ^##P < 0.01 vs. PKC-δ–overexpressed cells at 5.5 mM glucose; ⁺⁺P < 0.01 vs. control at 16.5 mM glucose; ^¶¶P < 0.01 vs. PKC-δ–overexpressed cells at 16.5 mM glucose. These results were derived from three different cloned populations of SMC overexpressing PKC-δ.