Figure 10.

DOP receptor dephosphorylation is mediated by PP1. (A–C) HEK293 cells stably expressing HA-hDOP receptor were transfected with siRNA targeting either (A) a combination of PP1α, PP1β and PP1γ (PP1-Mix), or (B) PP1α, PP1β, or PP1γ, or non-silencing siRNA control (SCR) for 72 h. After stimulation with 1 µM DPDPE for 10 min at 37 °C, cells were washed three times with cold citrate buffer and then incubated in serum-free medium in the absence of DPDPE for indicated times at 37 °C. Lysates were immunoblotted with antibody to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Knock down of protein expression by siRNA was confirmed by Western blot blot (bottom panels in A,B). Densitometry readings, above the blots, were normalized to those in SCR-transfected cells, which were set to 100%. Data are mean ± SEM from three to four independent experiments. *p < 0.05 vs. SCR by one-way ANOVA with Bonferroni’s post-hoc test.