Fig. 3.

RhoC regulates migration through ERK1/2. HUVECs were transfected with control or RhoC siRNA for 48 h, serum-starved overnight, and treated with VEGF-A for 5, 10, 15 or 20 min (+V5, +V10, +V5 and +V20, respectively). (A) Cell lysates were collected and immunoblotted (IB) with antibodies against phosphorylated ERK1/2 (pERK1/2), total ERK1/2, phosphorylated p38 MAPKs (pP38MAPK), phosphorylated Akt1, Akt and Akt3 (pAkt1/2/3), total Akt1, Akt and Akt3 (Akt1/2/3), phosphorylated JNK family proteins (pSAPK/JNK) and α-tubulin (loading control). (B) After serum starvation, cells were treated with 10 or 20 µM of MEK1 inhibitor for 1 h and 5×10⁴ cells were seeded into collagen-coated Transwell chambers and were then inserted into 24-well plates containing low-serum EGM. VEGF-A (10 ng/ml) was added in the lower chamber and a Transwell migration assay was performed for 4 h. Results are mean±s.d. (experiments were repeated at least three times in triplicates). *P≤0.05 and **P≤0.001 (paired two-tailed Student's t-test). (C) Cell lysates were collected and western blotted with antibodies against phosphorylated LIMK1/2 (pLIMK), total LIMK1, total LIMK2, phosphorylated MLC2 (pMLC-2), RhoC and β-actin (loading control). Vertical lines indicate where lanes were removed and composite images were generated from the same immunoblot. Please see supplementary material Fig. S3 for densitometry plots of the blots shown in A and C.