PKC-α but not PKC-β is the upstream signal in TNF-α-induced P115RhoGEF phosphorylation. (A) Serum-starved cells were transfected with vector-2, PKCα-shRNA, PKCβ-shRNA or pretreated with Gö6976. Serum-starved cells were then labeled with 32P for 4 h in phosphate-free medium. Cells were stimulated with TNF-α for 1 min, and then p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. The results show that depletion of PKC-α by Gö6976 or PKCα-ShRNA prevents the phosphorylation of p115RhoGEF in response to TNF-α. In contrast, depletion of PKC-β isozyme expression by using PKCβ-shRNA had no effect on P115RhoGEF phosphorylation. (B) Serum-starved cells were transfected with vector-1, n19RhoA, vector-2 and P115-shRNA. Cells were stimulated with TNF-α for 1 min, and then PKC-α activation was detected by vitro enzyme assasy. The results show that n19RhoA and P115-shRNA transfections had no effect on PKC-α activation. (n = 3 for each condition). *: p < 0.05 vs. Vector-2, △: p < 0.05 vs. Vector-1.-, absence; +, presence.