Fig. 2

PRDX6 is recruited to Gαi3 following norBNI treatment and regulates KOR/Gαi association. a Scatterplot of FLAG-Gαi3-interacting proteins, depicting the ratio of norBNI/vehicle in forward and reverse replicates. For experiment design, see Supplementary Fig. 2a; for individual proteins, see Supplementary Data 1. Proteins whose associations were changed by norBNI treatment in both replicates are indicated in red. b The increase in Gαi immunoprecipitated with myc after norBNI in mycKOR expressing cells was blocked by 10 µM MJ33 pretreatment (*, Student’s t-test, P < 0.05, n = 3–5). c NorBNI treatment (10 µM, 1 h) significantly increased phospho-JNK immunoreactivity in KORGFP expressing cells and was not blocked by 10 µM MJ33 pretreatment. (Significant effect of norBNI, **P < 0.01, but not MJ33 or interaction; two-way ANOVA, n = 9). d–f MycKOR expressing cells were immunostained for KOR and PRDX6. Colocalization was quantified by the Pearson correlation coefficient between intensity of KOR and PRDX6 immunoreactivity across 7–10 cells for each replicate. Representative images are shown in Supplementary Fig. 4a. Representative line plot profile analysis for a vehicle (d) and norBNI (10 µM, 1 h) (e) treated cell. Line plot is presented as the intensity normalized to the maximal intensity for that cell. f NorBNI treatment (significantly increased colocalization of KOR-IR and PRDX6-IR (*, paired t-test, P < 0.05, n = 8). No change after norBNI was observed when pretreated with 1 µM SP610025 (paired t-test, P = 0.6, n = 6). g NorBNI (10 µM, 1 h) significantly increased HA immunoreactivity in the membrane fraction of HEK293 cells stably expressing mycKOR and HA-PRDX6 ((P < 0.05) one sample t-test, n = 7). This increased was blocked by pretreatment with JNK-IN-8 (1 µM) (Student’s t-test, P < 0.05, n = 4). h Representative immunoblot for g. i HEK293 cells stably co-expressing mycKOR and HA-PRDX6 were treated with vehicle or norBNI (10 µM, 1 h) and lysed in PLA2 buffer. Whole cell, membrane, or cytosol fractions were collected and analyzed for PLA2 activity. NorBNI selectively increased PLA2 activity in the membrane fraction (& (P < 0.05) one sample t-test, n = 8). This increase was blocked by pretreatment with JNK-IN-8 (1 µM) or MJ33 (10 µM) (P < 0.05, one-way ANOVA; *P < 0.05 Holm–Sidak post hoc vs. vehicle + norBNI; n = 4-8). Error bars represent mean ± SEM