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. 2011 Jul 27;286(38):32931–32936. doi: 10.1074/jbc.M111.224899

FIGURE 6.

FIGURE 6.

Inhibition of sst2A dephosphorylation results in enhanced and prolonged ERK activation. A, HEK293 cells stably expressing the rat sst2A receptor were transfected with PP1β siRNA or a nonsilencing RNA (CTL) for 72 h. Where indicated, cells were incubated with 100 ng/ml pertussis toxin (PTX) 16 h before agonist exposure. Cells were then exposed to 1 μm SS-14 for 0, 5, or 10 min. The levels of phosphorylated ERK1/2 and total ERK1/2 were then determined by Western blot analysis. siRNA knockdown was confirmed using a PP1β antibody. Bands were quantified and expressed as percent ERK activation induced by SS-14 exposure for 5 min in CTL-transfected cells (dotted line). Data correspond to the mean ± S.E. (error bars) from four independent experiments performed in duplicate. Results were analyzed by one-way ANOVA followed by the Bonferroni post-test (*, p < 0.05). B, HEK293 cells stably expressing the rat sst2A receptor were transfected with PP1β siRNA or a nonsilencing RNA (CTL) for 72 h. Where indicated, cells were incubated with 100 ng/ml pertussis toxin (PTX) 16 h before agonist exposure. Cells were then exposed to 1 ng/ml EGF for 0, 5, or 10 min. The levels of phosphorylated ERK1/2 and total ERK1/2 were then determined by Western blot analysis. Bands were quantified and expressed as percent ERK activation induced by EGF exposure for 5 min in CTL-transfected cells (dotted line). Data correspond to the mean ± S.E. from three independent experiments performed in duplicate. C, HEK293 cells stably expressing the rat NK1receptor were transfected with PP1β siRNA or a nonsilencing RNA (CTL) for 72 h. Cells were then exposed to 1 μm substance P (SP) for 0, 5, or 10 min. The levels of phosphorylated ERK1/2 and total ERK1/2 were then determined by Western blot analysis. The experiment was repeated three times. Note that inhibition of PP1β expression resulted in an aberrantly enhanced and prolonged ERK activation in SS-14- and substance P- but not in EGF-treated cells. The positions of molecular mass markers are indicated on the left (in kDa).