Fig. 2.

Dexamethasone regulation of ROCK1 and ROCK2 kinase activity. Top Panel: Con8 cells were treated with or without dexamethasone for 3 days in the presence or absence of the Rho kinase inhibitor Y-27632. Cell lysates were incubated with ROCK1 polyclonal antibodies or IgG control antibodies, bound to Protein G sepharose beads. A kinase assay using the Rho kinase substrate MYPT1 was performed with the immunoprecipitated samples as described in Materials and Methods. The kinase assay mixture was electrophoretically separated on an SDS gel and subjected to Western blot analysis for both phospho-MYPT1 or total MYPT1. Middle panel: The experiment was performed as described for the top panel except that ROCK2 polyclonal antibodies were employed for the kinase-specific immunoprecipitations. Lower Panel: The relative ROCK1 and ROCK2 specific enzymatic activities were quantified as the level of detected phospho-MYPT1 in immunoprecipitates using the anti-ROCK antibodies and then subtracting the basal signal using the IgG control antibodies. The band intensities were quantified using densitometry, and the bar graphs represent the average of three independent experiments.