rabbit anti arrestin1 antibody  (Novus Biologicals)

Journal: Biology direct

Article Title: Nucleolin in the cell membrane promotes Ang II-mediated VSMC phenotypic switching by regulating the AT1R internalization function : Nucleolin promotes Ang II-mediated VSMC phenotypic switching.

doi: 10.1186/s13062-025-00615-0

Figure Lengend Snippet: Fig. 7 NCL promotes VSMC phenotypic switching in mice. A. RT-qPCR and WB analysis of NCL expression in mice. B. Blood pressure monitoring in mice. C. H&E staining of thoracic aorta from mice. Scale bar = 100 and 25 µM. D. WB analysis of NCL expression. E. IF staining for detecting Ki67 expression. Scale bar = 25 µM. F. WB analysis of β-arrestin1, β-arrestin2, Clathrin, AP-2, and Caveolae-1 expression. G. WB analysis of Rab4 and Rab11expression. H. IF assay for detecting α-SMA and OPN expression. Scale bar = 25 µM. I. WB analysis of AT1R, p-p38MAPK/p38MAPK, p-JNK/JNK, p-ERK/ERK, and p-PKCα/ PKCα expression. The mice were treated with Ang II and injected with sh-NC or sh-NCL lentiviruses in the tail vein. *p < 0.05 vs. sh-NC. *p < 0.05 vs. Sham, #p < 0.05 vs. Ang II + sh-NC. n = 5

Article Snippet: Primary antibody include NCL (ab136649, Abcam), AT1R (25343-1-AP, Proteintech), p38 (AWA00938, Abiowell), p-p38 (AWA48964, Abiowell), p-JNK (AWA45692, Abiowell), JNK (AWA45693, Abiowell), p-ERK (AWA01140, Abiowell), ERK (AWA01141, Abiowell), p-PKCα (AWA43973, Abiowell), PKCα (AWA43970, Abiowell), α-SMA (AWA00778, Abiowell), SM22α (AWA47343, Abiowell), OPN (AWA43353, Abiowell), β-arrestin1 (#47698, CST), β-arrestin2 (#3857, CST), Clathrin (AWA43132, Abiowell), AP-2 (AWA41820, Abiowell), Caveolae-1 (AWA47438, Abiowell), Rab4 (AWA00556, Abiowell), Rab11 (AWA46835, Abiowell), β-actin (AWA80002, Abiowell), and PCNA (AWA00425, Abiowell) were incubated at 4 °C overnight.

Techniques: Quantitative RT-PCR, Expressing, Staining, Injection

Journal: Journal of neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Fig. 3 β-arrestin1 in astrocytes modulates cognitive impairments and astrocytic reactivity in the mouse model for POD. A β-arrestin1 protein levels in brain lysate of WT and β-arrestin1−/− mice. B Experimental protocol and timeline of the POD mouse model. C Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. D Time (%) spent in the novel arm in the Y-maze test. E Bouts of novel arm entry in the Y-maze test. F Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. G Latency (s) to reach the hidden platform in the probe test of Morris water maze test. H Crossing times in target quadrant in the probe test of Morris water maze test. I Representative immunofluorescent staining of GFAP in the hippocampus. J Analysis of GFAP-positive cell body area in the hippocampus. K Analysis of GFAP-positive cell numbers in the hippocampus. L Heatmap of the expression level of the neurotoxic astrocytes-specific transcripts in the hippocampus. M Expression of C3, Serping1 and Psmb8 in the hippocampus. N Densitometric analysis of C3, Serping1 and Psmb8. O Schematic diagram of the mice model with micro-injection of AAV-siβ-arrestin1 into the hippocampus. P Immunofluorescent co-localization of GFP and GFAP (red) after the AAV micro-injection. Q Representative immunofluorescent staining of GFAP (red) in the hippocampus. R Analysis of astrocytic reactivity by GFAP-positive cell body area and GFAP-positive cell numbers in the hippocampus. S Bouts of novel arm entry in the Y-maze test. T Time (%) spent in the novel arm in the Y-maze test. U Latency (s) to reach the hidden platform in the probe test of Morris water maze test. V Crossing times in target quadrant in the probe test of Morris water maze test. For C-M except for R, data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the WT-CON group or NC AAV CON group. #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the WT-POD mice or NC AAV POD. For R, data are analyzed by unpaired Student’s t-test. *P < 0.05 and ***P < 0.001 vs. the NC AAV POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 7–10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris– HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-βarrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1- AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Microinjection, Western Blot

Journal: Journal of neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Fig. 4 β-arrestin1 deletion aggravates the neurotoxic reactivity of primary astrocytes. A Schematic of the experimental design. B Expression of C3, Serping1 and Psmb8 in the primary astrocytes. C Densitometric analysis of C3, Serping1 and Psmb8. D Heat map of A1 astrocytic genes in primary cell cultures. E Immunofluorescent staining of C3 (green) and GFAP (red) in primary astrocytes. F Immunofluorescent staining of Serping1 (red) and GFAP (green) in primary astrocytes. G Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between groups. H Relative co-localized signals of the GFAP-positive and Serping1-positive immunofluorescent particles between groups. I Astrocytes were stained with MitoSOX and analyzed by flow cytometry. J JC-1 staining in astrocytes were analyzed by flow cytometry. K Quantification of the mitochondrial ROS in MitoSOX staining. L Quantification of the loss of mitochondrial membrane potential in JC-1 staining measured by flow cytometry. M Oxygen consumption rates were evaluated by Seahorse. N Quantification of oxygen consumption for ATP production, basal respiration and proton leak. O ATP levels in astrocytes. Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the WT CON-MCM group. #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the WT LPS-MCM group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris– HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-βarrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1- AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Fig. 6 β-arrestin1-biased ligand induces the interaction of β-arrestin1 and Drp1 to inhibit mitochondrial fission in vitro. A Schematic of the experimental design. B Immunoblotting analysis of Drp1 proteins in cell lysates of primary astrocytes immunoprecipitated with β-arrestin1 antibody. C Immunoblotting analysis of β-arrestin1 proteins in cell lysates of primary astrocytes immunoprecipitated with Drp1 antibody. D Immunofluorescent staining of TOMM20 (green) and Drp1 (red) in primary astrocytes. E Relative co-localized signals of the TOMM20-positive and Drp1-positive immunofluorescent particles between groups. F Representative images of Mitotracker Green in primary astrocytes under confocal microscope. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. *P < 0.05 vs. the CON group. #P < 0.05 vs. the LPS-MCM group. Values are presented as means ± SEM from three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris– HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-βarrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1- AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: In Vitro, Western Blot, Immunoprecipitation, Staining, Microscopy

Journal: Journal of neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Fig. 8 β-arrestin1-biased ligand Carvedilol protects POD mice from pro-inflammatory phenotypes. A Experimental protocol and timeline of the mouse model. B Representative immunofluorescent staining of GFAP in the hippocampus. C Relative GFAP-positive cell body area in the hippocampus. D Relative GFAP-positive cell numbers in the hippocampus. E Heatmap of the expression level of the A1-specific transcripts in hippocampal samples. F Expression of C3, Serping1 and Psmb8 in the hippocampus. G Densitometric analysis of C3, Serping1 and Psmb8. H Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. I Time (%) spent in the novel arm in the Y-maze test. J Bouts of novel arm entry in the Y-maze test. K Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. L Latency (s) to reach the hidden platform in the probe test of Morris water maze test. M Crossing times in target quadrant in the probe test of Morris water maze test. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the CON group. #P < 0.05, ##P < 0.01 and ###P < 0.01 vs. the POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris– HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-βarrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1- AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Western Blot

Journal: Journal of neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium.

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Fig. 10 Proposed working model of postoperative delirium triggered by astroglial β-arrestin1 deletion. Decreased β-arrestin1 in postoperative delirium induced astrocytic reactivity to facilitate the pathological progression. β-Arrestin1 deletion triggered the disassociation between with β-arrestin1 and cytoplasmic Drp1 to promote mitochondrial translocation of Drp1 and cause excessive mitochondrial fragmentation and mitochondrial dysfunctions. Activation of β-arrestin1-biased signaling by agonist recovers neurotoxic astrocytic reactivity and phenotypes of postoperative delirium mouse model

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris– HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-βarrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1- AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Translocation Assay, Activation Assay