Fig. 5 A. (C) Quantification of the level of PKA in
Fig. 5 A. (D) Immunoblots showed the inhibitory effects of 3A5C7 mAb on morphine-induced upregulation of p-MOR Serine 375 and PKA in SH-SY5Y cells. (E) Quantification of the level of p-MOR in
Fig. 5 D. (F) Quantification of the level of PKA in
Fig. 5 D. (G) The effects of 3A5C7 mAb on the level of intracellular cyclic adenosine monophosphate (cAMP) in HEK293T-MOR cells. (H) The effects of 3A5C7 mAb on the level of intracellular cAMP in SH-SY5Y cells. Cells were subjected to morphine and 3A5C7 antibody for 72 h, then treated with forskolin (Fs, 10 μM) for 30 min at 37 °C. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine the concentration of cAMP. Forskolin-stimulated cAMP level was used as the basal value. (I) The influences of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in intracellular cAMP in HEK293T-MOR cells. (J) The influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in intracellular cAMP in SH-SY5Y cells. (K) Immunoblots showing the influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in PKA in HEK293T-MOR cells. (L, M) Quantification of the relative expression levels of PKA in
Fig. 5 K. (N) Immunoblots showing the influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in PKA in SH-SY5Y cells. (O, P) Quantification of the relative expression levels of PKA in
Fig. 5 N. [ 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. NIg: normal IgG; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC: negative blank control; si-GRK2: small interfering RNA (siRNA) for GRK2; si-β-arrestin2: siRNA for β-arrestin2; si-NC: control siRNA. " width="100%" height="100%">
Journal: Journal of Pharmaceutical Analysis
Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis
doi: 10.1016/j.jpha.2023.06.008
Figure Lengend Snippet: 3A5C7 monoclonal antibody (mAb) attenuates morphine tolerance in vitro. (A) Immunoblots showed the inhibitory effects of 3A5C7 mAb on morphine-induced upregulation of phospho-MOR (p-MOR) Serine 375 and protein kinase A (PKA) in HEK293T-MOR cells. (B) Quantification of the level of p-MOR in Fig. 5 A. (C) Quantification of the level of PKA in Fig. 5 A. (D) Immunoblots showed the inhibitory effects of 3A5C7 mAb on morphine-induced upregulation of p-MOR Serine 375 and PKA in SH-SY5Y cells. (E) Quantification of the level of p-MOR in Fig. 5 D. (F) Quantification of the level of PKA in Fig. 5 D. (G) The effects of 3A5C7 mAb on the level of intracellular cyclic adenosine monophosphate (cAMP) in HEK293T-MOR cells. (H) The effects of 3A5C7 mAb on the level of intracellular cAMP in SH-SY5Y cells. Cells were subjected to morphine and 3A5C7 antibody for 72 h, then treated with forskolin (Fs, 10 μM) for 30 min at 37 °C. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine the concentration of cAMP. Forskolin-stimulated cAMP level was used as the basal value. (I) The influences of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in intracellular cAMP in HEK293T-MOR cells. (J) The influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in intracellular cAMP in SH-SY5Y cells. (K) Immunoblots showing the influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in PKA in HEK293T-MOR cells. (L, M) Quantification of the relative expression levels of PKA in Fig. 5 K. (N) Immunoblots showing the influences of GRK2 and β-arrestin2 knockdown on the inhibitory effects of 3A5C7 on morphine-induced increase in PKA in SH-SY5Y cells. (O, P) Quantification of the relative expression levels of PKA in Fig. 5 N. [ 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. NIg: normal IgG; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC: negative blank control; si-GRK2: small interfering RNA (siRNA) for GRK2; si-β-arrestin2: siRNA for β-arrestin2; si-NC: control siRNA.
Article Snippet: The rabbit polyclonal anti-phospho-MOR Serine 375 (p-MOR) antibody (#3,451), rabbit polyclonal anti-protein kinase A (PKA) antibody (#4,782), and rabbit polyclonal anti-tubulin antibody (#2,146) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Small Interfering RNA
