FIGURE 3.

Both Rho kinase isoforms ROCK1 and ROCK2 phosphorylate MYPT-1 and MLC and can compensate for the loss of the other. HUVECs were incubated with 50 nm οf either a nonspecific control siRNA (NC) or a ROCK1-specific, ROCK2-specific, or dual ROCK1/ROCK2 siRNAs for 24 h in a transfection medium. The medium was then replaced with standard growth medium (EGM-2), and cells were incubated for an additional 24 h. Cells were then serum-starved overnight prior to LPA exposure for 5 min. A, cell viability was assessed for each treatment with CellTiter-Glo luminescent cell viability assay (Promega) following the manufacturer's instructions. Values are means (n = 4) ± S.E. B, efficiency of gene knockdown was assessed by Western blotting with ROCK1- and ROCK2-specific antibodies (top two panels). Phosphorylation of MYPT-1 and MLC was assessed by Western blotting with phosphospecific antibodies. Total MYPT-1, total MLC, and GAPDH were used as normalization controls.