integrin β 3 antibody  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: MFG-E8-Derived Oligopeptide MOP3 Facilitates Anti-Inflammatory M2-like Macrophage Polarization in Gut Ischemia/Reperfusion

doi: 10.3390/cells15070606

Figure Lengend Snippet: MOP3 increases M2 activity and decreases M1 activity in an α v β 3 -dependent manner. Peritoneal macrophages from adult mice were treated with PBS or eCIRP (1 µg/mL) and MOP3 (10 µg/mL). Groups treated with eCIRP and MOP3 were pretreated with either IgG or α v β 3 antibody. Supernatant was collected at 24 h and measured for ( A ) IL-10 and ( B ) TNFα by ELISA. Data are expressed as mean ± SEM. Results were tested for normality by Shapiro–Wilk test and QQ plots. Results were evaluated by ANOVA and Tukey’s multiple-comparisons test (* p < 0.05 vs. PBS, # p < 0.05 vs. IgG).

Article Snippet: For cytokine detection experiments, separate groups of eCIRP and MOP3-treated cells were pretreated with IgG (10 μg/mL; bs-0295p; Bioss, Woburn, MA, USA) or α v β 3 polyclonal ab (10 μg/mL; cat. bs-1310r; Bioss).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: MFG-E8-Derived Oligopeptide MOP3 Facilitates Anti-Inflammatory M2-like Macrophage Polarization in Gut Ischemia/Reperfusion

doi: 10.3390/cells15070606

Figure Lengend Snippet: MOP3 clearance of eCIRP promotes M2 polarization of macrophages in mesenteric ischemia/reperfusion injury. MOP3 facilitates phagocytosis of eCIRP by macrophages via α v β 3 integrin, which leads to an increase in polarization toward M2 relative to M1. This phenomenon is demonstrated in mesenteric ischemia/reperfusion, improving healing while decreasing inflammation.

Article Snippet: For cytokine detection experiments, separate groups of eCIRP and MOP3-treated cells were pretreated with IgG (10 μg/mL; bs-0295p; Bioss, Woburn, MA, USA) or α v β 3 polyclonal ab (10 μg/mL; cat. bs-1310r; Bioss).

Techniques:

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a IP 1 production in HEK293 cells transfected with either control siRNA or integrin β 3 siRNA, along with the expressing of vector and G qi9 , or GABA B receptor and G qi9 , under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, not significant (ns) > 0.05. b Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c –f Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion ( c ), without (control) or with shear stress ( c ), Blebbistatin (50 μM, 30 min) treatment ( d ), RGDS (10 μM, 12 h) treatment ( e ), or CGP54626 (50 μM, 30 min) treatment ( f ). Blots are representative from at least three biologically independent experiments ( b , n = 4; c , suspension or adhesion, n = 3; control or with shear stress, n = 4; d , n = 3; e, n = 3; f , n = 4). The amount of integrin β 3 immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in ( e ) and analyzed using unpaired t test (two-tailed) to determine significance. ** P < 0.01. g Schematic representation of the BRET assay detecting direct interaction between GABA B receptor and integrin β 3 . Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1 Rluc or GB2 Rluc ) as luminescence donor. Venus was fused in C-terminal of integrin β 3 or integrin α V subunit (integrin β 3 Venus or integrin α V Venus ) as fluorescence acceptor. h , i Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V ( h ), or GB2 and integrin β 3 ( i ) detected using BRET titration assay. The BRET between mGlu2 with Rluc fused in the C-terminal and 5HT2a receptor with Venus fused in the C-terminal was measured as positive control. The BRET between GB1 Rluc and Venus was measured as negative control. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments (upper to lower, h , left, n = 16, 10, 12; right, n = 5, 5; i , n = 9, 9) each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.

Article Snippet: Integrin β 3 antibody (#sc-46655, 1:100) and anti-mouse IgG (#sc-2025, 1:500) for the co-immunoprecipitation experiment were purchased from Santa Cruz Biotechnology (Shanghai, China).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Positive Control, Negative Control, Binding Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic representation of GABA B -ΔVFT truncation, in which GB1 subunit lacks of the VFT domain (GB1-ΔVFT), but retains the ability to be activated by positive allosteric modulator Rac BHFF (yellow square). Co-immunoprecipitation experiments were performed using anti-HA antibody targeting to the HA tag, which is fused in the N-terminal of GB1 or GB1-ΔVFT. b Co-immunoprecipitation of GABA B receptor or GABA B -ΔVFT and integrin β 3 using anti-HA antibody. Blots are from one representative of five biologically independent experiments. c IP 1 production in cells transfected with GABA B receptor, or GABA B -ΔVFT, along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -ΔVFT and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which Rac BHFF was added for 200 seconds. Data are present as mean ± s.e.m. from 135 cells recorded.

Article Snippet: Integrin β 3 antibody (#sc-46655, 1:100) and anti-mouse IgG (#sc-2025, 1:500) for the co-immunoprecipitation experiment were purchased from Santa Cruz Biotechnology (Shanghai, China).

Techniques: Immunoprecipitation, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: Antagonist CGP54626 -bound GABA B receptor fails to interact with integrin β 3 , whereas integrin β 3 interacts with GB1 VFT of GABA B receptor with constitutive activity. Mechanical force promotes GB1 VFT and integrin β 3 interaction to stabilize the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact, further inducing an allosteric re-arrangement of the GABA B receptor 7TM towards an TM6-TM6 active conformation, culminating in the asymmetric activation of the receptor with G protein under GB2. Mechanical force acts as a positive allosteric modulator to boost up GABA-induced GABA B receptor activation through interaction between GB1 VFT and integrin β 3 .

Article Snippet: Integrin β 3 antibody (#sc-46655, 1:100) and anti-mouse IgG (#sc-2025, 1:500) for the co-immunoprecipitation experiment were purchased from Santa Cruz Biotechnology (Shanghai, China).

Techniques: Activity Assay, Activation Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a IP 1 production in HEK293 cells transfected with either control siRNA or integrin β 3 siRNA, along with the expressing of vector and G qi9 , or GABA B receptor and G qi9 , under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, not significant (ns) > 0.05. b Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c –f Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion ( c ), without (control) or with shear stress ( c ), Blebbistatin (50 μM, 30 min) treatment ( d ), RGDS (10 μM, 12 h) treatment ( e ), or CGP54626 (50 μM, 30 min) treatment ( f ). Blots are representative from at least three biologically independent experiments ( b , n = 4; c , suspension or adhesion, n = 3; control or with shear stress, n = 4; d , n = 3; e, n = 3; f , n = 4). The amount of integrin β 3 immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in ( e ) and analyzed using unpaired t test (two-tailed) to determine significance. ** P < 0.01. g Schematic representation of the BRET assay detecting direct interaction between GABA B receptor and integrin β 3 . Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1 Rluc or GB2 Rluc ) as luminescence donor. Venus was fused in C-terminal of integrin β 3 or integrin α V subunit (integrin β 3 Venus or integrin α V Venus ) as fluorescence acceptor. h , i Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V ( h ), or GB2 and integrin β 3 ( i ) detected using BRET titration assay. The BRET between mGlu2 with Rluc fused in the C-terminal and 5HT2a receptor with Venus fused in the C-terminal was measured as positive control. The BRET between GB1 Rluc and Venus was measured as negative control. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments (upper to lower, h , left, n = 16, 10, 12; right, n = 5, 5; i , n = 9, 9) each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.

Article Snippet: Integrin β 3 antibody (#13166, 1:1000), β-actin (#4967, 1:1000), phospho-Myosin Light Chain 2 (MLC-PP, #3675, 1:1000), GFAP (#3670, 1:1000), anti-mouse IgG HRP-linked antibody (#7076, 1:10000), anti-rabbit IgG HRP-linked antibody (#7074, 1:10000), anti-rabbit IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5151, 1:10000) and anti-mouse IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5257, 1:10000) were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Positive Control, Negative Control, Binding Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic representation of GABA B -ΔVFT truncation, in which GB1 subunit lacks of the VFT domain (GB1-ΔVFT), but retains the ability to be activated by positive allosteric modulator Rac BHFF (yellow square). Co-immunoprecipitation experiments were performed using anti-HA antibody targeting to the HA tag, which is fused in the N-terminal of GB1 or GB1-ΔVFT. b Co-immunoprecipitation of GABA B receptor or GABA B -ΔVFT and integrin β 3 using anti-HA antibody. Blots are from one representative of five biologically independent experiments. c IP 1 production in cells transfected with GABA B receptor, or GABA B -ΔVFT, along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -ΔVFT and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which Rac BHFF was added for 200 seconds. Data are present as mean ± s.e.m. from 135 cells recorded.

Article Snippet: Integrin β 3 antibody (#13166, 1:1000), β-actin (#4967, 1:1000), phospho-Myosin Light Chain 2 (MLC-PP, #3675, 1:1000), GFAP (#3670, 1:1000), anti-mouse IgG HRP-linked antibody (#7076, 1:10000), anti-rabbit IgG HRP-linked antibody (#7074, 1:10000), anti-rabbit IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5151, 1:10000) and anti-mouse IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5257, 1:10000) were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Immunoprecipitation, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: Antagonist CGP54626 -bound GABA B receptor fails to interact with integrin β 3 , whereas integrin β 3 interacts with GB1 VFT of GABA B receptor with constitutive activity. Mechanical force promotes GB1 VFT and integrin β 3 interaction to stabilize the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact, further inducing an allosteric re-arrangement of the GABA B receptor 7TM towards an TM6-TM6 active conformation, culminating in the asymmetric activation of the receptor with G protein under GB2. Mechanical force acts as a positive allosteric modulator to boost up GABA-induced GABA B receptor activation through interaction between GB1 VFT and integrin β 3 .

Article Snippet: Integrin β 3 antibody (#13166, 1:1000), β-actin (#4967, 1:1000), phospho-Myosin Light Chain 2 (MLC-PP, #3675, 1:1000), GFAP (#3670, 1:1000), anti-mouse IgG HRP-linked antibody (#7076, 1:10000), anti-rabbit IgG HRP-linked antibody (#7074, 1:10000), anti-rabbit IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5151, 1:10000) and anti-mouse IgG (H + L) (DyLight TM 800 4X PEG Conjugate) (#5257, 1:10000) were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Activity Assay, Activation Assay