ROCK (Rho kinase) assay

Cells were fixed and harvested in 10% TCA containing 10 mM DDT. Pellets were dissolved in 10 µl of 1 M Tris base and mixed with 100 µl of extraction buffer (8 M urea, 2% SDS, 5% sucrose, and 5% 2-mercaptoethanol). Equal amounts of protein from each cell extract were subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with an antibody specific for phospho-myosin binding subunit (phospho-Thr853-MYPT1) or myosin binding subunit (MYPT1), and bands were visualized by enhanced chemiluminescence. ROCK activity was expressed as the ratio of phospho-MYPT1 to total MYPT1.