Figure 9.

Inhibition of DOP receptor dephosphorylation by calyculin A but not okadaic acid. (A,B) HEK293 cells stably expressing HA-hDOP receptor were either preincubated with the indicated concentrations of (A) calyculin A or (B) okadaic acid for 30 min at 37 °C and then stimulated with 1 µM DPDPE for 10 min at 37 °C. Subsequently, cells were washed three times with ice cold citrate buffer and then incubated in serum-free medium in the absence of DPDPE for 0, 20 or 40 min in the presence of the above indicated concentrations of (A) calyculin A or (B) okadaic acid at 37 °C. Lysates were immunoblotted with antibody to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Densitometry readings, above the blots, were normalized to those in DPDPE-stimulated cells (0 nM and 0 min), which were set to 100%. Data are mean ± SEM from three independent experiments. *p < 0.05 vs. DPDPE controls by one-way ANOVA with Bonferroni’s post-hoc test.