VEGF-C activated RhoA/ROCK-2 pathway. (A-B) RhoA expression and its activity were assayed in cells treated with VEGF-C (100 ng/mL) for 24 or 48 h alone, or treated with VEGF-C for 48 h in the presence of Flt-4/IgG (100 ng/mL). Active, GTP-bound RhoA was immunoprecipitated with Rhoteckin and subsequently assayed with western analysis with an anti-RhoA Ab. ** = P < 0.01 vs. corresponding control; *** = P < 0.001 vs. corresponding control; ## = P < 0.01 vs VEGF-C 48 h. (C-D) ROCK-2 expression and its activity were assayed in cells treated as indicated. ROCK-2 was immunoprecipitated with a specific Ab and the IPs were used to phosphorylate the bait protein, myelin basic protein (MBP). ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). ** = P < 0.01 vs. corresponding control; ## = P < 0.01 vs VEGF-C 48 h. All the above experiments were performed in triplicates and representative images are shown.