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. 2019 Oct 1;294(45):16884–16896. doi: 10.1074/jbc.RA119.009592

Figure 2.

Figure 2.

Arrestin-independent and -dependent U50,488 stimulated phospho-JNK occur by distinct mechanisms. A–D, HEK293 cells stably expressing KORGFP were treated transfected with control siRNA (si-ctl), siRNA against arrestin 2 (si-arr2), or siRNA against arrestin 3 (si-arr3) 48 h prior to treatment with vehicle or 10 μm U50,488 for 15 or 60 min. Cell lysates were then immunoblotted. A and B, knockdown of arrestin (arr) 2 and 3 was confirmed by immunoblotting. Representative immunoblots (A) and quantification (B) are shown (two-way ANOVA (significant effect of siRNA, F2,26 = 2.711, p = 0.0023, and significant interaction between siRNA and arrestin isoform, F2,26, p < 0.0001; n = 4–7; arrestin expression, F1,26 = 0.003077, p = 0.9562) with Holm–Šidák post hoc comparison against control siRNA (**, p = 0.0015; ***, p = 0.0002)). C and D, cell lysates were immunoblotted for phospho-JNK. Representative immunoblots (C) and quantification (D) are shown. Transfection with siRNA against arrestin 3 significantly blocked JNK phosphorylation after 15-min U50,488 treatment (two-way ANOVA (significant effect of arrestin, F2,44 = 3.312, p = 0.0457, n = 6–10; effect of treatment time, F1,44 = 1.703, p = 0.1987; effect of interaction, F2,44 = 2.227, p = 0.1199) with Holm–Šidák post hoc comparison against control siRNA (*, p = 0.0277)). E–G, HEK293 cells stably expressing KORGFP were pretreated with 10 μm Y27632 (Y27), 100 μm NSC23766 (NSC), 5 μm Gö6976, or vehicle (veh) minutes prior to 15-min 10 μm U50,488 (U50) treatment. Representative immunoblots (E and F) and quantification (G) are shown. Pretreatment with NSC23766 or Gö6976, but not Y27632, significantly blocked stimulation of phospho-JNK by U50,488 at 15 min (one-way ANOVA (F3,46 = 7.026, p = 0.0005, n = 8–19) with Holm–Šidák post hoc comparison against vehicle pretreatment (**, p = 0.0089)). H–J, cells were treated as for E–G but treated with U50,488 for 60 min. Representative immunoblots (H and I) and quantification (J) are shown. Treatment with Y27632 or NSC23766 significantly blocked stimulation of phospho-JNK by U50,488 at 60 min, whereas pretreatment with Gö6976 increased stimulation of phospho-JNK by U50,488 at 60 min (one-way ANOVA (F3,37 = 11.58, p < 0.0001, n = 8–16) with Holm–Šidák post hoc comparison against vehicle pretreatment (*, p = 0.0319; **, p = 0.0043)). Graphs depict mean ± S.E. with individual replicates shown.