GRK2 and arrestin2 regulate UTP-mediated arterial contraction 201 Figure 6 Depletion of endogenous arrestin2 attenuates P2Y2-receptor desensitization. MSMCs were nucleofected with NC, anti-arrestin2 or anti- arrestin3 siRNAs (100 nM) (see Section 2). After 48 h, cells were lysed and 40 mg of protein loaded for SDS – PAGE separation and immunoblotting. (A) Representative immunoblot showing arrestin2 depletion (upper panel), and the same blot is shown after longer exposure to highlight arrestin3 (lower panel, lower band) depletion, shown in: non-transfected cells (lane 1), or cells treated with 100 nM of anti-arrestin2 (lane 2), anti-arrestin3 (lane 3), or NC (lane 4) siRNAs. (B) Cumulative densitometric data show mean arrestin expression + SEM from four animal cell preparations, **P,0.01 vs. NC siRNA (one-way ANOVA; Dunnett’s post hoc test). To assess the effects of arrestin depletion on UTP- or ET1-stimulated PLC signalling, MSMCs were nucleofected with 0.5 mg eGFP-PH and with 100 nM of either NC, anti-arrestin2 or anti-arrestin3 siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/Rmax/R2 (for UTP) or R1/R1 (for ET-1) desensitization protocols (see Section 2). Representative traces from single cells transfected with control siRNA (C and F ), anti-arrestin2 (D), or anti-arrestin3 (G). Traces (C ) and (D) show UTP responses, while (F ) and (G) show ET-1 responses. Cumulative data are shown as means + SEM from 5 to 13 cells from preparations from three to four separate animals for UTP- (E) and ET1-stimulated cells (H ). Statistical signiﬁcance is indicated as *P , 0.05 or **P , 0.01 vs. NC siRNA-treated MSMCs (one-way ANOVA; Dunnett’s post hoc test).