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

Fig. 3.

Fig. 3

Effects of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2 knockdown on the endocytosis of mu-opioid receptor (MOR) induced by morphine and 3A5C7 monoclonal antibody (mAb). (A) Immunofluorescence staining showed that the MOR endocytosis induced by morphine and 3A5C7 mAb was reduced in HEK293T-MOR cells with GRK2 or β-arrestin2 knockdown. (B, C) Flow cytometry indicated that MOR endocytosis induced by morphine and 3A5C7 mAb was reduced in HEK293T-MOR cells with GRK2 (B) or β-arrestin2 knockdown (C). (D, E) Flow cytometry indicated that MOR endocytosis induced by morphine and 3A5C7 mAb was reduced in SH-SY5Y cells with GRK2 (D) or β-arrestin2 (E) knockdown. (F) Immunoblots showed that GRK2 knockdown reduced MOR endocytosis and translocation of β-arrestin2 from cytoplasm to cell membrane in HEK293T-MOR cells. (G) Quantification of the relative levels of MOR and β-arrestin2 in the membrane and cytoplasm from Fig. 3F. (H) Immunoblots showed that GRK2 knockdown reduced MOR endocytosis and translocation of β-arrestin2 from cytoplasm to cell membrane in SH-SY5Y cells. (I) Quantification of the relative levels of MOR and β-arrestin2 in the membrane and cytoplasm from Fig. 3H. HEK293T-MOR and SH-SY5Y cells were transfected with small interfering ribonucleic acid (siRNA) for GRK2 (si-GRK2), β-arrestin2 (si-β-arrestin2), or control siRNA (si-NC) for 48 h, co-cultured with morphine and 3A5C7 mAb/normal IgG (NIg) for another 72 h, then cell membrane and plasma proteins were extracted and subjected to Western blot analysis. (J) Immunoblots showed that downregulation of β-arrestin2 reduced endocytosis of MOR induced by morphine and 3A5C7 mAb in HEK293T-MOR cells. (K) Quantification of the relative levels of MOR in the membrane and cytoplasm from Fig. 3J. (L) Immunoblots showed that downregulation of β-arrestin2 reduced endocytosis of MOR induced by morphine and 3A5C7 mAb in SH-SY5Y cells. (M) Quantification of the relative levels of MOR in the membrane and cytoplasm from Fig. 3L. 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. 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. DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride.