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. 2012 Feb 28;287(16):12736–12749. doi: 10.1074/jbc.M111.314617

FIGURE 3.

FIGURE 3.

Effect of GRK2 knockdown on MOP receptor phosphorylation and desensitization. A, heterologous phosphorylation. Left panel, representative Western blots showing the levels of GRK2, actin, and MOP receptor Ser-377 phosphorylation (P-MOP) in cells transfected with transfection agent alone (0), negative control siRNA (si−), or GRK2 siRNAs (1127 and 1128) and treated or not with 1 μm 1DMe for 30 min. Right panel, histogram showing the quantification of Ser-377 phosphorylation induced by 1 μm 1DMe in cells transfected with transfection agent alone (0), negative control siRNA (si−), or GRK2 siRNAs (1127 and 1128). Data are expressed as percent of stimulation over nonstimulated cells (means ± S.E. of three independent experiments). **, p < 0.01; one-way ANOVA followed by Bonferroni post-tests. B, homologous phosphorylation. Representative Western blot showing the levels of GRK2, actin, and MOP receptor Ser-377 phosphorylation (P-MOP) in cells transfected with negative control siRNA (si−) or GRK2 siRNAs (1127 and 1128) and treated or not with 1 μm DAMGO for 30 min. C, heterologous desensitization. Inhibition of potassium-evoked calcium influx by 30 s of treatment with 0.1 μm DAMGO and effect of pretreatment with 1 μm 1DMe for 30 min in control cells and cells transfected with control (si−) or GRK2 (si1128) siRNAs. Results are expressed as percent of the DAMGO effect in control cells (c). Intracellular calcium was measured using Fluo-4. ***, p < 0.001 versus control (c); #, p < 0.05 versus 1DMe; one-way ANOVA followed by Bonferroni post-tests. Number of cells: 48 (c), 47 (1DMe), 60 (C si1128), 58 (1DMe si1128), and 41 (1DMe si−).