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

FIGURE 7.

FIGURE 7.

U69,593 and 6′-GNTI have divergent signaling effects on ERK1/2 in mouse primary striatal neurons. Primary striatal neurons were serum-starved for 1 h prior to the determination of ERK1/2 phosphorylation (10 μm for 10 min). Representative images of 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 is provided (mean ± S.E.). A, WT neurons were pretreated with either vehicle (Veh, 0.9% saline) or nor-BNI (Nor, 1 μm) during the last 15 min of the serum starvation. U69,593 (U69) activates ERK1/2 in striatal neurons in a nor-BNI-sensitive manner (one-way ANOVA: for U69,593, F(2,18) = 9.550, p = 0.0015; Bonferroni's post hoc test: vehicle versus U69,593, **, p < 0.01, U69,593 versus nor-BNI + U69,593, ^^, p < 0.01). In contrast, 6′-GNTI (6′) represses ERK1/2 activation in striatal neurons, and this repression is not sensitive to nor-BNI (one-way ANOVA: for 6′-GNTI, F(2,19) = 9.527, p = 0.0014; Bonferroni's post hoc test: vehicle versus drug treatment groups, **, p < 0.01; n = 5–6 independent neuronal preparations). In a separate set of experiments, the neurons were treated with 6′-GNTI for 2 min or nor-BNI (1 μm) alone for 10 min. 6′-GNTI-mediated activation of ERK1/2 is not merely shifted, as a 2-min treatment does not shift phosphorylation levels away from basal (Student's t test: p > 0.05). Nor-BNI treatment alone also represses ERK1/2 phosphorylation levels (Student's t test: *, p < 0.05; n = 4 independent neuronal preparations). B, U69,593-mediated ERK1/2 activation in WT striatal neurons is not sensitive to overnight pretreatment with pertussis toxin (PTX; 100 ng/ml) (Student's t test: vehicle versus U69,593 within each pretreatment group, *, p < 0.05; n = 3 independent neuronal preparations). Representative images are from different parts of the same gel. C, neither U69,593 nor 6′-GNTI induces an increase in ERK1/2 phosphorylation levels in striatal neurons cultured from βarr2-KO mice (Student's t test: p > 0.05; n = 4 independent neuronal preparations).