Fig. 7.

3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance via G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2 pathway in mice. (A) Immunoblot showing that GRK2 was knocked-down by short hairpin ribonucleic acid (shRNA) in dorsal root ganglions (DRGs). (B) Quantification of the protein level of GRK2 in Fig. 7A. (C) Immunoblot showing that β-arrestin2 was knocked-down by shRNA in DRGs. (D) Quantification of the protein level of β-arrestin2 in Fig. 7C. (E) Hotplate tests demonstrating the effects of GRK2 or β-arrestin2 knockdown on the anti-tolerance efficacy of mAb 3A5C7. Two-way analysis of variance (ANOVA) with Bonferroni's post hoc tests were used for statistical analysis. ^∗∗P < 0.01 and ^∗∗∗∗P < 0.0001, sh-NC + morphine + mAb group vs. sh-NC + morphine group; ^###P < 0.001 and ^####P < 0.0001, sh-NC + morphine + mAb group vs. sh-GRK2 + morphine + mAb group; ^&P < 0.05 and ^&&&P < 0.001, sh-NC + morphine + mAb group vs. sh-β-arrestin2 + morphine + mAb group (n = 5 mice in each group). (F) Representative immunofluorescence images of MOR and Rab5 for each treatment in mouse DRGs (n = 3 mice in each group). (G) The protein levels of hippocampal protein kinase A (PKA) from mice in each treatment group. (H, I) Quantification of the relative protein levels of PKA from Fig. 7G. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ^∗∗P < 0.01 and ^∗∗∗P < 0.001. (J) Inhibitory effects of acute 3A5C7 mAb administration on naloxone-precipitated withdrawal jumping were reduced in GRK2- and β-arrestin2-knockdown mice. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ^∗∗∗∗P < 0.0001 vs. sh-NC + morphine group; ^####P < 0.0001 vs. sh-NC + morphine + mAb group (n = 5 mice in each group). Data were presented as the mean ± standard error of mean. sh-NC: control shRNA; sh-GRK2: shRNA for GRK2; sh-β-arrestin2: shRNA for β-arrestin2; MPE: maximum possible effect; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.