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: Fig. 2. 3A5C7 monoclonal antibody (mAb) regulated morphine-induced mu-opioid receptor (MOR) endocytosis through a G protein-coupled receptor kinase 2 (GRK2)/b-arrestin2- dependent way. (A) Western blot and quantification showed the upregulation of GRK2 in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Western blot and

Article Snippet: Anti-MOR antibody (AOR-011; Alomone Labs), anti-GRK2 antibody, and anti-b-arrestin2 antibody were used.

Techniques: Western Blot

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: Fig. 3. Effects of G protein-coupled receptor kinase 2 (GRK2) and b-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 b-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 b-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 b-arrestin2 (E) knockdown. (F) Immunoblots showed that GRK2 knockdown reduced MOR endocytosis and translocation of b-arrestin2 from cytoplasm to cell membrane in HEK293T-MOR cells. (G) Quantification of the relative levels of MOR and b-arrestin2 in the membrane and cytoplasm from Fig. 3F. (H) Immunoblots showed that GRK2 knockdown reduced MOR endocytosis and translocation of b-arrestin2 from cytoplasm to cell membrane in SH-SY5Y cells. (I) Quantification of the relative levels of MOR and b-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), b-arrestin2 (si-b- 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 b-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 b-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. Naþ-Kþ adenosine triphosphatase (ATPase) a1 subunit was used as internal reference of cell membrane and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as

Article Snippet: Anti-MOR antibody (AOR-011; Alomone Labs), anti-GRK2 antibody, and anti-b-arrestin2 antibody were used.

Techniques: Knockdown, Staining, Flow Cytometry, Western Blot, Translocation Assay, Membrane, Transfection, Control, Cell Culture, Clinical Proteomics

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: Fig. 7. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance via G protein-coupled receptor kinase 2 (GRK2)/b-arrestin2 pathway in mice. (A) Immunoblot showing that GRK2 was knocked-down by short hairpin ribonucleic acid (shRNA) in dorsal root ganglions (DRGs). (B) Quantification of the protein level of GRK2 in Fig. 7A. (C) Immunoblot showing that b-arrestin2 was knocked-down by shRNA in DRGs. (D) Quantification of the protein level of b-arrestin2 in Fig. 7C. (E) Hotplate tests demonstrating the effects of GRK2 or b-arrestin2 knockdown on the anti-tolerance efficacy of mAb 3A5C7. Two-way analysis of variance (ANOVA) with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ****P < 0.0001, sh-NC þ morphine þ mAb group vs. sh-NC þ morphine group; ###P < 0.001 and ####P < 0.0001, sh- NC þ morphine þ mAb group vs. sh-GRK2 þ morphine þ mAb group; &P < 0.05 and &&&P < 0.001, sh-NC þ morphine þ mAb group vs. sh-b-arrestin2 þ morphine þ mAb group (n ¼ 5 mice in each group). (F) Representative immunofluorescence images of MOR and Rab5 for each treatment in mouse DRGs (n ¼ 3 mice in each group). (G) The protein levels of hippocampal protein kinase A (PKA) from mice in each treatment group. (H, I) Quantification of the relative protein levels of PKA from Fig. 7G. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ***P < 0.001. (J) Inhibitory effects of acute 3A5C7 mAb administration on naloxone-precipitated withdrawal jumping were reduced in GRK2- and b-arrestin2-knockdown mice. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ****P < 0.0001 vs. sh-NC þ morphine group; ####P < 0.0001 vs. sh-NC þ morphine þ mAb group (n ¼ 5 mice in each group). Data were presented as the mean ± standard error of mean. sh-NC: control shRNA; sh-GRK2: shRNA for GRK2; sh-b-arrestin2: shRNA for b-arrestin2; MPE: maximum possible effect; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Anti-MOR antibody (AOR-011; Alomone Labs), anti-GRK2 antibody, and anti-b-arrestin2 antibody were used.

Techniques: Western Blot, shRNA, Knockdown, Control

Journal: eLife

Article Title: Two-photon imaging in mice shows striosomes and matrix have overlapping but differential reinforcement-related responses

doi: 10.7554/elife.32353

Figure Lengend Snippet: Figure 1. Striosomes are labeled with tdTomato in Mash1-CreER;Ai14 mice that received tamoxifen at E11.5. Images illustrate two examples (rows) of striosomal labeling of cell bodies and neuropil by tdTomato (A,D, red) as verified by MOR1 immunostaining identifying striosomes (B,E, blue). Merged images show overlap of tdTomato and MOR1 labeling (C,F). Scale bars indicate 100 mm. DOI: https://doi.org/10.7554/eLife.32353.002 The following figure supplement is available for figure 1:

Article Snippet: For staining, sections were first rinsed 3 5 min in PBS-Tx (0.01 M PBS + 0.2% Triton X-100), then were incubated in blocking buffer (Perkin Elmer TSA Kit) for 20 min followed by incubation with primary antibodies for GFP (Polyclonal, chicken, Abcam ab13970, 1:2000) and MOR1 (Poly- clonal, goat, Santa Cruz sc-7488, 1:500).

Techniques: Labeling, Immunostaining

Journal: Frontiers in Immunology

Article Title: False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases

doi: 10.3389/fimmu.2021.666114

Figure Lengend Snippet: Description of the SARS-CoV-2 serological assays and the test codes used in this study.

Article Snippet: H , InTec Products inc., Haicang Xiamen, China , INTEC_ Colloidal Gold (whole blood/Serum/Plasma) Rapid SARS-CoV-2 Antibody (IgM/IgG) , Nucleocapsid protein ( ) , ITP16001-TC25 , Combined IgM+IgG: 98%.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery