Fig. 1.

Effects of 3A5C7 monoclonal antibody (mAb) on morphine-induced mu-opioid receptor (MOR) endocytosis from cell membrane to cytoplasm. (A) Immunofluorescence staining indicated the endocytosis of MOR in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Immunofluorescence staining indicated the endocytosis of MOR in SH-SY5Y cells treated with morphine and 3A5C7 mAb. (C) Immunofluorescence staining manifested the colocalization of MOR and Rab5 in HEK293T-MOR cells subjected to 3A5C7 mAb and morphine. (D, E) Flow cytometry showed the endocytosis of MOR in HEK293T-MOR cells co-treated with morphine and 3A5C7 mAb. (F, G) Flow cytometry showed the endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immunoblots showed the endocytosis of MOR in HEK293T-MOR cells co-treated with morphine and 3A5C7 mAb. 3A5C7 mAb (10 μg/mL) and morphine (10 μM) were simultaneously added to the culture media for 72 h before assay. (I, J) Quantification of relative MOR level in the cell membrane (I) and cytoplasm (J) from Fig. 1H. (K) Immunoblots showed the endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. 3A5C7 mAb (10 μg/mL) and morphine (10 μM) were simultaneously added to the culture media for 72 h before assay. (L, M) Quantification of relative MOR level in the cell membrane (L) and cytoplasm (M) from Fig. 1K. 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 in Figs. 1H−M. [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, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001. ns: no significance. NC: negative blank control; NIg: normal IgG; DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride.