ROCK1 interacts with PTEN. (A) WT and ROCK1^−/− BMMs cultured for 10 days in the presence of M-CSF were starved overnight and stimulated with 100 ng/mL M-CSF for 5 minutes. Equal amount of lysates were subjected to immunoprecipitation with an anti-PTEN antibody, and Western blot analysis was performed using an anti-ROCK1 antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated ROCK1^−/− BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively. The arrow indicates the interaction of ROCK1 with PTEN in WT BMMs stimulated with M-CSF. (B-C) Enhanced phosphorylation of AKT and GSK-3β in ROCK1^−/−-deficient BMMs. WT and ROCK1^−/− BMMs were starved for 24 hours and stimulated with 100 ng/mL M-CSF for 5 minutes. Equal amounts of lysates were subjected to Western blot analysis using an anti-phospho AKT or phospho GSK3-β antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated ROCK1^−/− BMMs, respectively (n = 3). (D) Increased expression of cyclin D1 in ROCK1^−/− BMMs. WT and ROCK1^−/− BMMs were harvested and lysed in lysis buffer. Equal amounts of protein lysates were subjected to Western blot analysis using an antibody against cyclin D1. The top arrow indicates the expression of cyclin D1 in WT and ROCK1^−/− BMMs. The bottom arrow indicates β-actin expression in each lane (n = 3).