Fig. 3

NorBNI and morphine induce ROS production via JNK and PRDX6. MycKOR a, c or mycMOR b, d expressing HEK293 cells were pretreated with vehicle, AACOCF3 (10 µM), JNK-IN-8 (1 µM), SP610025 (1 µM), MJ33 (10 µM), or N-acetyl-cysteine (NAC, 10 µM) 30 min prior to being treated for 1 h with norBNI (10 µM), morphine (10 µM), fentanyl (10 µM) or naloxone (10 µM). a, b Cells were imaged for ROS using CellROX Green; scale bar represents 12.5 µm. c Quantification of ROS in mycKOR cells. NorBNI, but not naloxone, increased CellROX Green fluorescence; this was blocked by pretreatment with MJ33, JNK-IN-8, SP610025, or NAC, but not AACOCF3 (one-way ANOVA, P < 0.0001, n = 3-11; *,^# P < 0.05, **P < 0.01, Holm–Sidak post hoc analysis). d Quantification of ROS in mycMOR cells. Morphine, but not fentanyl or norBNI, increased CellROX Green fluorescence; this increase was blocked by SP610025 (one-way ANOVA, P < 0.001, n = 4–5; *P < 0.05, **P < 0.01, Holm–Sidak post hoc analysis). e Schematic illustrating the experimental protocol used to generate the results shown in f, g, and in Supplementary Fig. 5. HEK293 cells stably co-expressing mycKOR and FLAG-GΑI3 were pretreated with vehicle, SP610025, or MJ33 and treated 5.5 h with norBNI prior to harvest. Cell membranes were extracted in the presence of N-ethylmaleimide (50 mM) to covalently bind unmodified cysteines, and immunoprecipitated with anti-myc to isolate mycKOR and mycKOR-associated FLAG-Gαi. Myc immunoprecipitates were treated with hydroxylamine to cleave palmitic acids and then incubated with BMCC-biotin to biotinylate previously palmitoylated cysteines. Palmitoylation was measured by probing with streptavidin and normalizing to anti-Gαi immunoreactivity. f NorBNI significantly reduced palmitoylation of Gαi coimmunoprecipitated with mycKOR, but not Gαi that was not KOR-associated (^& P < 0.05, one sample t-test with correction for multiple comparisons; n = 5–6); this change in palmitoylation was blocked by MJ33 pretreatment (*P < 0.05 Student’s t-test with Welch’s correction). g Representative immunoblots from f. Error bars represent mean ± SEM