Cells were lysed in RIPA buffer in the presence of proteinase inhibitor cocktail. Then, the total protein extracts were separated and transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against ROCK1 and GAPDH (Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4°C and then blotted for 1 hour at room temperature with an appropriate secondary antibody, followed by enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Little Chalfont, UK).
Small RNA Northern blot analysis was performed as previously described.23 (link) Band intensities were measured using Quantity One image analysis software (Bio-Rad, Hercules, CA, USA). The RNA Northern Blot Assay Kit for miR-199a was purchased from BioCat GmbH (Heidelberg, Germany) (Cat No MP-0080-SO).
Small RNA Northern blot analysis was performed as previously described.23 (link) Band intensities were measured using Quantity One image analysis software (Bio-Rad, Hercules, CA, USA). The RNA Northern Blot Assay Kit for miR-199a was purchased from BioCat GmbH (Heidelberg, Germany) (Cat No MP-0080-SO).