Fig. 5.

Combined effects of GRK5 or -6 siRNA and H89-mediated inhibition on the kinetics of ERK activation by the V2R. (A) Cells were transfected with the FLAG-V2R-encoding plasmid and the indicated siRNAs. Cells were preincubated for 15 min with DMSO or 20 μM H89 and stimulated with 100 nM dvdAVP at 37°C for the indicated periods. Equal amounts of protein from each sample were used to visualize the phosphorylation of ERK (Left) and total ERK (Right) by immunoblotting. (B and C) Signals were quantified by densitometry and expressed as percentage of the maximal phosphorylated ERK obtained at 5 min of stimulation in control (CTL) siRNA-transfected cells. Each data point represents the mean ± SEM from at least four independent experiments. ***, P < 0.001 compared with entire control curve.