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. 2023 Jun 20;13(10):1135–1152. doi: 10.1016/j.jpha.2023.06.008

Fig. 2.

Fig. 2

3A5C7 monoclonal antibody (mAb) regulated morphine-induced mu-opioid receptor (MOR) endocytosis through a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent way. (A) Western blot and quantification showed the upregulation of GRK2 in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Western blot and quantification showed the upregulation of GRK2 in SH-SY5Y cells treated with morphine and 3A5C7 mAb. (C) Immunofluorescence staining showed the translocation of β-arrestin2 from cytoplasm to cell membrane in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (D) Immunoblots showed the translocation of β-arrestin2 from cytoplasm to cell membrane in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (E, F) Quantification of relative β-arrestin2 level in the cell membrane (E) and cytoplasm (F) from Fig. 2D. (G) Immunoblots showed the translocation of β-arrestin2 from cytoplasm to cell membrane in SH-SY5Y cells treated with morphine and 3A5C7 mAb. (H, I) Quantification of relative β-arrestin2 level in the cell membrane (H) and cytoplasm (I) from Fig. 2G. (J) Coimmunoprecipitation (Co-IP) assay demonstrated the interaction of MOR with GRK2 and β-arrestin2 after treatment with morphine and 3A5C7 mAb in HEK293T-MOR cells. (K, L) Quantification of the relative levels of GRK2 and β-arrestin2 interacting with MOR from Fig. 2J. (M) Co-IP assay demonstrated the interaction of MOR with GRK2 and β-arrestin2 after treatment with morphine and 3A5C7 mAb in SH-SY5Y cells. (N, O) Quantification of the relative levels of GRK2 and β-arrestin2 interacting with MOR from Fig. 2M. Na+-K+ adenosine triphosphatase (ATPase) α1 subunit was used as internal reference of cell membrane and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal reference of cytoplasm. [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) was used as positive controls. One-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis. Data were presented as mean ± standard error of mean (n = 3 independent experiments). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. NC: negative blank control; NIg: normal IgG; DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride.