anti mor polyclonal antibody  (Novus Biologicals)

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: Frontiers in Molecular Neuroscience

Article Title: Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice

doi: 10.3389/fnmol.2023.1139118

Figure Lengend Snippet: Altered transcriptome of D1- and D2-MSN, over-expression of GAD65, and modification of the striosome/matrix balance in the striatum of Shank3 Δ11/Δ11 mice. (A) Enrichment of RNAseq DEGs in the striatum of Shank3 Δ11/Δ11 mice compared to cell-specific gene clusters suggested by and . Genes under-expressed in Shank3 Δ11/Δ11 striatum are enriched in D1-MSN clusters while genes over-expressed in Shank3 Δ11/Δ11 striatum are enriched in D2-MSN clusters. Ratio genes correspond to the proportion of DEGs enriched in the cluster. Over-representation analyses were performed with Fisher’s hypergeometric tests and p -values were adjusted for multiple testing with the Benjamini–Hochberg procedure within each category of DEGs. (B) Comparison of the log of the fold change of gene expression of selected genes in Shank3 Δ11/Δ11 vs. Shank3 +/+ mice (LogFC RNAseq, Y axis) and the β values (slope of the linear regression) for low self-grooming vs. high self-grooming ( β value grooming, X axis). (C,D) Confocal images of striatum coronal sections of one-year old Shank3 +/+ male mice. (C) GAD65 immunoreactivity (red) is enriched in striatal microzones identified as striosomes by immunostaining for μ-opioid receptor (MOR, in green), a canonical marker of striosomes. (D) Increased GAD65 immunoreactivity in striosomes (arrows) and subcallosal streak (two-headed arrows) compared to surrounding matrix is observed with two different antibodies: the rabbit polyclonal antibody (Invitrogen PA5-77983 in red) used in (C,E,F) and a mouse monoclonal antibody (Millipore MAB351, in green). (E,F) Distribution and quantification of GAD65 immunoreactivity in dorsal striatum sections of a Shank3 +/+ (E) and a Shank3 Δ11/Δ11 (F) mouse (images generated by stitching multiple maximum-intensity projected z-stacks). The arrows point to striosomes and the arrowheads to subcallosal streak. In the dorsal striatum ROI (surrounded in green), the unlabeled myelinated fibers are colored in red, the regions of higher expression of GAD65 (striosomes) are colored in green, and the regions of lower expression of GAD65 (matrix) are uncolored. The cortex ROI used as a reference is surrounded in red. Note that the acquisition parameters were adjusted for each brain hemi-section in order to have no saturating signal for GAD65 in the striatal ROI. Increased intensity in the striatum thus leads to a staining which appears weaker in the cortex. (G–I) Comparison of GAD65 immunoreactivity in the striosome and matrix compartments of the dorsal striatum in 5 Shank3 +/+ (green) and 9 Shank3 Δ11/Δ11 (orange) 1-year old male mice. (G) Relative surface of the GAD65-enriched striosome compartment (surface of striosomes/surface of (striosomes + matrix). (H) Relative GAD65 labeling intensity in the striosomal compartment of the striatum compared to cortex. (I) Relative GAD65 labeling intensity in the matrix compartment of the striatum compared to cortex. Data, generated from analysis of 9–14 images per animal, are presented as box-plots (median, first, and third quartiles).

Article Snippet: The free-floating sections were rinsed three times in PBS (pH 7.4) and then incubated in NH4Cl (Sigma-Aldrich) 50 mM in PBS for 15 min. After three PBS washes (5 min each), sections were incubated for 1 h at room temperature in PBS containing 1% bovine serum albumin (Applichem) and 0.3% Triton X-100 (Sigma-Aldrich) (PBS/BSA/TX) before incubation for ≈ 20 h at room temperature with the following primary antibodies diluted in PBS/BSA/TX: rabbit polyclonal anti-GAD65 (Invitrogen PA5-77983, 1/200), mouse monoclonal anti-GAD65 (Millipore MAB351, 1/500), guinea pig polyclonal anti MOR (Millipore AB5509, 1/100), rabbit polyclonal anti-VGLUT1 (Synaptic Systems 135303, 1/1000), and mouse monoclonal anti-SHANK3 (Santa Cruz Biotechnology sc-377088, 1/1000).

Techniques: Over Expression, Modification, Expressing, Immunostaining, Marker, Generated, Staining, Labeling