Fig. 4.

Effects of 3A5C7 monoclonal antibody (mAb) on mu-opioid receptor (MOR) recycling from cytoplasm to cell membrane. (A) Immunofluorescence staining of HEK293T-MOR cells to demonstrate the recycling of MOR. (B) Immunofluorescence staining of SH-SY5Y cells to demonstrate the recycling of MOR. (C, D) Flow cytometry demonstrating the recycling of MOR in HEK293T-MOR cells (C) and corresponding quantification as percentages of control cell surface MOR without any treatment (D). (E, F) Flow cytometry demonstrating the recycling of MOR in SH-SY5Y cells (E) and corresponding quantification as percentages of control cell surface MOR without any treatment (F). HEK293T-MOR or SH-SY5Y cells were exposed to morphine (10 μM) and 3A5C7 (10 μg/mL) for 1 h to induce internalization which were washed out thereafter. MOR recycling was monitored at 0 min, 15 min, 30 min, and 1 h after washout. (G) Western blot showed the MOR recycling from cytoplasm to cell membrane. HEK293T-MOR or SH-SY5Y cells were treated as indicated and cell membrane and plasma proteins were extracted for assays. (H) Immunofluorescence staining showed the relationship between internalized MOR and lysosome associated membrane protein-1 (LAMP-1), the marker of lysosome. Cells were treated with morphine and 3A5C7 mAb for 1 h at 37 °C before assay. 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. Student's t 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 < 0.0001 versus recycle 0 min in Figs. 4D and F. DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride; NC: negative blank control.