RLC phosphorylation in response to anti-β2GPI antibodies is prevented by MLCK or ROCK inhibition. Endothelial cells were incubated with β2GPI and anti-β2GPI antibodies in the absence or presence of increasing concentrations of the MLCK inhibitor ML-7 or the ROCK inhibitor Y-27632. (A) Lysates were immunoblotted with anti-phosphoRLC (T18/S19) (upper and middle panels) or anti-RLC (total protein). (B) Endothelial cells were treated with scrambled (Sc) RNA (control) or siRNA specific for ROCK-1, ROCK-2, or MLCK; blotting with anti-actin antibodies was used to demonstrate equal loading. (C) Effect of siRNA knockdown of ROCK-1, ROCK-2, or MLCK on levels of phosphorylated RLC. Significant inhibition was caused by knockdown of each of the kinases.
