Protein kinase C-α and p115RhoGEF regulate TNF-α-induced RhoA activation. (A) Bend.3 cells were pretreated with or without Gö6976 (1 μM), a selective inhibitor of conventional PKC isozymes. RhoA activation was assayed after 5 min of TNF-α challenge. The results show that Gö6976 pretreatment of Bend.3 cells blocked TNF-α-induced RhoA activation. Bend.3 cells were transfected with vector-2(empty PLKO.1-puro vector), PKCα-shRNA, PKCβ-shRNA or p115-shRNA in the presence or absence of TNF-α. RhoA activation was partially prevented in PKCα-shRNA and P115-shRNA transfected cells, whereas RhoA activation did not affect PKCβ-shRNA transfected cells. (B) The Bend.3 cells transfected with vector-2, PKCα-shRNA, PKCβ-shRNA and p115-shRNA respectively. Stably transfected cells were used for experiments after selection by Puromycin (300 ug/ml). The inhibitory levels of p115RhoGEF (upper), PKC-α (middle) and PKC-β (bottom) expression were confirmed by western blot. (C) The time course of PKC-α and RhoA activation after TNF-α (10 ng/ml) treatment was detected. The results showed that TNF-α-induces rapid activation of PKC-α as well as RhoA at 0.5 min. (n = 3 for each condition). *: p < 0.05 vs. Vector-2, △: p < 0.05 vs. untreated Bend.3 cells. -, absence; +, presence.