Reduced phosphorylation of MYPT1 in ROCK1^−/− macrophages. (A) WT BMMs were starved overnight and stimulated with 100 ng/mL M-CSF for 5 minutes. Equal amounts of lysates were subjected to immunoprecipitation with an anti-ROCK1 antibody. Rho kinase activity was initiated by adding MYPT1 as a substrate for Western blot analysis using an anti-phospho MYPT1 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. The top arrow indicates phospho MYPT1 levels in WT and ROCK1^−/− BMMs on stimulation with M-CSF; the bottom arrow indicates β-actin levels in WT and ROCK1^−/− BMMs in unstimulated and stimulated conditions. The bar graph on top indicates the density of each band as analyzed by NIH Image software. The bar graph is presented as intensity of pMYPT1 in arbitrary units in WT and ROCK1^−/− BMMs (n = 3). (B) Enhanced PIP₃ levels in ROCK1^−/− BMMs. WT and ROCK1^−/− BMMs were starved in serum-free medium for 24 hours and then labeled with [³²P] orthophosphate (0.5 mCi/mL) for 2 hours. Cells were then stimulated with M-CSF (100 ng/mL) for 5 minutes before harvesting. Phospholipids were extracted and analyzed on a thin layer chromatography plate. Arrow indicates radiolabeled PIP₃ in WT and ROCK1^−/− BMMs. Lanes 1 and 2 indicate WT and ROCK1^−/− BMMs, respectively, stimulated with M-CSF. Lanes 3 and 4 indicate WT and ROCK1^−/− BMMs, respectively, unstimulated with M-CSF. The bar graph on top shows quantification of PIP₃ levels in WT and ROCK1^−/− BMMs. Amount of radioactive labeled ³²P incorporated in PIP₃ was quantified with ImageJ software, and PIP₃ levels are shown as arbitrary units.