Anti rock1 h 85 sc 5560

Cells were lysed with phospho-protein extraction buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EGTA, 1 mM NaF, supplemented with protease-phosphatase cocktail inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Equivalent amounts of total cell lysates were separated by 7.5% SDS-PAGE under denaturating conditions and transferred onto nitrocellulose membrane. Membranes were incubated overnight with the following primary antibodies: rabbit polyclonal anti-ROCK1 (H-85, sc-5560; Santa Cruz Biotechnology, Inc., San Diego, CA, USA) (1:1,000), goat polyclonal anti-ROCK2 (C-20, sc-1851; Santa Cruz Biotechnology) (1:1,000), mouse monoclonal anti-actin (C-4, MAB1501; Chemicon International, Inc., Temecula, CA, USA) (1:100,000). Donkey anti-rabbit (NA934; GE Healthcare, Piscataway, NJ, USA), donkey anti-goat (sc-2020; Santa Cruz Biotechnology) or sheep anti-mouse (NA931; GE Healthcare) horseradish peroxidase-linked secondary antibodies were employed and the signal was revealed by ECL western blotting detection reagents (EuroClone). Densitometric analysis was performed using GS-800 Imaging Densitometer and Quantity One 4.6.9 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).