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.

Dexamethasone-regulated expression of ROCK1 and ROCK2 protein in Con8 mammary tumor cells. Top Panel: Con8 cells were grown to 100% confluence, and treated with or without dexamethasone for 5 days. Western blots of electrophoretically fractionated cell extracts were probed with antibodies against ROCK1 and ROCK2, respectively, and actin as a loading control. Bar Graphs: The relative production of ROCK1 and ROCK2 protein was quantified as the detected levels of ROCK1 and ROCK2 proteins in comparison to the detected actin protein at a significantly shorter film exposure. The band intensities were quantified using densitometry, and the bars graphs represent the average of three independent experiments.

Inhibition of Rho kinase activity disrupts the glucocorticoid-induced and Rnd3/RhoE mediated tight junction sealing. Con8 mammary tumor cells or a Con8-derived cell line that stably expresses Rnd3/RhoE (C8-Rnd3 cells) were cultured in the presence or absence of 1 μM dexamethasone (Dex) for 4 days and then each cell culture treated with or without 10 μM Y-27632, a Rho kinase inhibitor. The monolayer transepithelial electrical resistance (TER) was monitored to examine tight junction sealing. The results are an average of three independent experiments.