Fig. 4. Bidirectional control of RhoA activity through V2 and EP₃ receptor stimulation in IMCD cells. IMCD cells were incubated as indicated in the legend to Fig. 1. Subsequently, GTP-bound RhoA was precipitated and detected by western blotting (for details see Materials and Methods). (A) Cells were either left untreated, incubated with AVP (100 nM; 15 minutes) or with C. difficile toxin B, which inhibits all proteins of the Rho family (4 µg/ml; 4 hours). (B) Cells were either left untreated, incubated with AVP or with both sulprostone and SC19220 in the absence or presence of AVP. (C) For quantification, immunoblots were densitometrically analyzed (for details see Materials and Methods). RhoA activity is indicated by the amount of GTP-bound RhoA related to the amount of total RhoA in the whole cell lysates. Values represent amounts of RhoA normalized to untreated control cells. Results are means ± s.e. of at least three independent experiments per condition (n=3-6). *, statistically different from untreated control cells (for untreated control cells versus sulprostone-treated cells P<0.01, for all other comparisons P<0.001); #, statistically different from AVP-stimulated cells (for AVP-stimulated cells versus + AVP + sulprostone P<0.05, for all other comparisons P<0.001).

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