Cells were lysed in phosphate-buffered saline (PBS) and protease inhibitor cocktail (Roche Diagnostics, Risch-Rotkreuz, Switzerland), Halt phosphatase inhibitor cocktail (Pierce, Rockford, IL, USA), and lysis buffer containing 0.5% NP-40, 0.5% deoxycholate, 150 mM sodium chloride, and 50 mM tris-HCL (pH 7.4). All lysates were subjected to a 15,871g spin for 5 min to remove nuclei and debris. Protein concentration was determined by bicinchoninic acid method (Pierce). Immunoblots were performed using standard procedures as described previously (74). A quantity of 50 μg of protein per sample was loaded per lane. Tubulin was used as a loading control. Images were captured using an Odyssey Image Station (Li-Cor), and band intensities were quantified using Odyssey Application Software Version 3.0 (Li-Cor). Primary antibodies were incubated overnight at 4°C. Primary antibodies include ROCK1 (Abcam, 45171), ROCK2 (Santa Cruz Biotechnology, catalog no. 5561), LIMK (Cell Signaling Technology, product no. 3842S), phospho-LIMK (Cell Signaling Technology, product no. 3841), phospho-cofilin (Cell Signaling Technology, product no. 3313), cofilin (Cell Signaling Technology, no. 3318), and tubulin (Iowa Hybridoma Bank). Secondary antibodies include Alexa Fluor 680 goat anti-rabbit (A21109, Life Technologies) and goat anti-mouse (Li-Cor, product no. 926-32210).

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