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
Journal: Life
Article Title: Loss of Corticostriatal Mu-Opioid Receptors in α-Synuclein Transgenic Mouse Brains
doi: 10.3390/life12010063
Figure Lengend Snippet: Lower MOR levels in corticostriatal WMT of A53T α-Syn tg mouse brains. ( A ). A lower magnification of a coronal mouse brain section, stained with Luxol Fast blue, showing the striatum. CC, corpus callosum; LV, lateral ventricle; CPu, caudate putamen. WMTs are shown as dark spots over the lightly stained striatal grey matter in the CPu. Bar = 0.5 mm. ( B ). A semi-thick A53T α-Syn mouse brain section (1 μm), stained with methylene blue, showing cross-sectioned axons in a WMT. Bar = 10 μm. ( C ). Paraffin-embedded coronal sections (6 μm) of α-Syn-/- (C57BL/6JOlaHsd) and A53T α-Syn mouse brains at 2 months of age, containing the dorsal striatum (representative images). Sections immunoreacted either with anti-MOR, anti-NF-200, anti-MBP, or anti calbindin 28 (CB-28) antibodies. The consecutive section of the α-Syn-/- brain were stained with the corresponding secondary antibody as a control (no 1°). Bar = 50 μm. ( D ). The relative WMTs immunoreactive signal of MOR or MBP normalized to the signal obtained for NF-200. Total CB-28 signal in striatal matrix normalized to NF200 signal. Mean ± SD of A53T mice at 2, 8, and 12 months of age. The horizontal bar represents the corresponding age-matched control mice, set at 100%. n = 4–5 brains for each genotype and each age group; 3–6 fields per brain; t -test with Bonferroni correction, * p < 0.017.
Article Snippet: Blots were immunoreacted with a polyclonal rabbit anti MOR ab (1:5000 Millipore, Rosh-Ha’ayin, Israel) or polyclonal rabbit anti-Calbindin-D-28-K (CB-28, 1:5000, Sigma, Rehovot, Israel).
Techniques: Staining, Control
Journal: Life
Article Title: Loss of Corticostriatal Mu-Opioid Receptors in α-Synuclein Transgenic Mouse Brains
doi: 10.3390/life12010063
Figure Lengend Snippet: MOR levels are lower in the striatum of Thy-1 WT α-Syn tg mouse brains. ( A ). Samples of membrane fractions P10 or P25, or supernatant S25 (30 μg protein) obtained from tissue punches containing the striatum of Thy-1 α-Syn and control mouse brains at 2 and 10 months of age analyzed by Western blotting and immunoreacted with anti-MOR or anti-Na+/K+ ATPase antibodies. The original western blot images are provided in the (mouse brain samples at 2 months of age) and (mouse brain samples at 10 months of age). ( B ). Graph showing quantitation of blots obtained in ( A ) mean ± SD of n = 4 mice. *, <0.05, t -test. ( C ). Western blot as in ( A ), the soluble S25 fraction immunoreacted with anti-CB-28 or anti β-actin antibodies. The original western blot images are provided in the (left side shows mouse brain samples at 2 months of age; the right side shows mouse brain samples at 10 months of age).
Article Snippet: Blots were immunoreacted with a polyclonal rabbit anti MOR ab (1:5000 Millipore, Rosh-Ha’ayin, Israel) or polyclonal rabbit anti-Calbindin-D-28-K (CB-28, 1:5000, Sigma, Rehovot, Israel).
Techniques: Membrane, Control, Western Blot, Quantitation Assay
Journal: Cell
Article Title: A Genetically Defined Compartmentalized Striatal Direct Pathway for Negative Reinforcement
doi: 10.1016/j.cell.2020.08.032
Figure Lengend Snippet: KEY RESOURCES TABLE
Article Snippet: Rabbit polyclonal anti-MOR , Immunostar , 24216.
Techniques: Virus, Software
Millipore is a verified supplier 