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. 2013 Jun 17;288(31):22387–22398. doi: 10.1074/jbc.M113.476234

FIGURE 4.

FIGURE 4.

6′-GNTI-stimulated ERK1/2 phosphorylation in CHO-KOR cells is pertussis toxin-sensitive. CHO-KOR cells were serum-starved for 1 h prior to drug treatment and the determination of ERK1/2 phosphorylation. Representative images of plates or Western blots and densitometric analyses are provided. For each sample, phosphorylated ERK1/2 (p-ERK1/2) was first normalized to total ERK1/2 (t-ERK1/2) and the -fold stimulation over vehicle or basal is provided (mean ± S.E.). A, following treatment with increasing concentrations of drug for 10 min, -fold stimulation was determined by immunocytochemistry in a 384-well plate format (In-cell Western). 6′-GNTI induces ERK1/2 phosphorylation to a similar extent as U69,593 (two-way ANOVA for drug: F(1,108) = 0.07, p = 0.7919; for concentration: F(7,108) = 79.85, p < 0.0001). 5′-GNTI does not induce ERK1/2 phosphorylation (n = 9 (U69,593 and 6′-GNTI) and n = 5 (5′-GNTI) performed in replicates of six). B, both U69,593-induced (U69, 10 μm for 10 min) and 6′-GNTI-induced (6′, 10 μm for 10 min) ERK1/2 phosphorylation are sensitive to pretreatment with nor-BNI (Nor; 10 μm for 15 min) as determined by Western blot analysis (one-way ANOVA: F(5,41) = 79.49, p < 0.0001; Bonferroni's post hoc test: vehicle versus drug treatment, ***, p < 0.001, U69,593 versus nor-BNI+U69,593, ^^^, p < 0.001, 6′-GNTI versus nor-BNI+6′-GNTI, ###, p < 0.001; n = 4 independent experiments). C, time course analysis of the 10 μm concentrations of U69,593 and 6′-GNTI reveals that ERK1/2 phosphorylation levels remain elevated for up to 2 h post-treatment with either ligand (one-way ANOVA: for U69,593, F(5,27) = 7.886, p < 0.0001; for 6′-GNTI, F(5,27) = 9.619, p < 0.0001; Bonferroni's post hoc test: basal versus U69,593, *, p < 0.05, **, p < 0.01, ***, p < 0.001, basal versus 6′-GNTI, ^, p < 0.05, ^^^, p < 0.001). U69,593 and 6′-GNTI do not induce any significant differences between their time courses of activation (two-way ANOVA for drug: F(1,54) = 3.17, p = 0.0805; for time: F(5,54) = 16.65, p < 0.0001; n = 4 independent experiments). D, U69,593- and 6′-GNTI-induced (10 μm for 10 min) ERK1/2 phosphorylation can be blocked by an overnight pretreatment (100 ng/ml) with pertussis toxin (PTX) (Student's t test: vehicle (veh) versus drug treatment, ***, p < 0.001; n = 3 independent experiments).