integrin β 3  (Proteintech)

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: eBioMedicine

Article Title: Parvimonas micra exacerbates periodontitis by infiltrating host cells through TmpC and circumventing lysosomal elimination via AppA

doi: 10.1016/j.ebiom.2026.106187

Figure Lengend Snippet: P. micra interacts with PDLSCs through TmpC-integrin α v β 3 axis. ( A ) Schematic representation of His pull-down. ( B ) Co-IP assay showing the interaction between purified His-TmpC and integrin α v β 3 of PDLSCs. ( C ) Sensorgrams of integrin α v β 3 binding to immobilised TmpC and K D value. ( D ) The interaction between TmpC (blue), integrin α v (green) and integrin β 3 (red), as predicted by molecular docking. ( E–F ) Bacterial attachment assay revealing altered bacterial recovery rate in PDLSCs treated with siIntegrin α v+ β 3 ( E ) or Cyclo ( F ). ( G–H ) Flow cytometry showing the percentages of invaded PDLSCs treated with siIntegrin α v+ β 3 ( G ) or Cyclo ( H ). ( I ) Knockdown of integrin α v β 3 altered downstream pFAK and the activation of NF-κB and ERK1/2 signalling pathways triggered by P. micra . β-actin was set as control. ( J ) Western blot showing altered activation of NF-κB and ERK1/2 signalling pathways triggered by P. micra in PDLSCs pretreated with Cyclo. β-actin was set as control. ( K ) Representative image and quantification of ALP staining in Cyclo-pretreated PDLSCs infected with P. micra WT or P. micra ΔtmpC . ( L ) Representative image and quantification of ARS staining of PDLSCs with blocked TmpC-integrin α v β 3 interaction. ( M ) Western blot of Col1, ALPL, Runx2 in Cyclo-pretreated PDLSCs infected with P. micra WT or P. micra ΔtmpC . GAPDH was set as control. ( N ) Flow cytometric analysis of FDAA-labelled P. micra internalisation in Cyclo-pretreated PDLSCs. ( O ) Schematic diagram showing the experimental design and timeline of rat models infected with P. micra and/or treated with Cyclo. ( P ) Quantitative analyses of CEJ-ABC, BV/TV, BMD and Tb.Sp. of the alveolar bone. Data are represented as the mean ± SD of three independent experiments ( n = 3) in (E–N) and six independent experiments ( n = 6) in (P). P values were determined by two-tailed unpaired Student's t -test in (E–H) and one-way ANOVA test in (I–N, P). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Article Snippet: Commercially available integrin α v β 3 (R&D Systems, 3050-AV-050) was diluted in sodium acetate (pH 5.0) and passed over the sensor chip in various concentrations from 0 μM to 160 μM, with the 5 μM concentration as internal control.

Techniques: Co-Immunoprecipitation Assay, Purification, Binding Assay, Flow Cytometry, Knockdown, Activation Assay, Control, Western Blot, Staining, Infection, Two Tailed Test

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: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

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: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

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: a Immunofluorescent staining of GFAP (red), GFP (green) and DAPI (cyan) in astrocytes transfected with control siRNA or GABA B receptor siRNA along with GFP, treatment with or without shear stress (15 dyn/cm 2 , 30 min). Images are representative from three biologically independent experiments. Scale bar: 10 μm. b , c Analysis of the cell size and GFAP expression of astrocytes with the same treatment in ( a ). Measurements are made on each cell by cell basis (ROI) from three biologically independent experiments (number of cells from left to right: n = 47, 37, 32, 40). Data are present as mean ± s.e.m and analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test to determine significance. *** P < 0.001, ** P < 0.01, ns: non-significant. d RNA interference efficacy of the GB1 in astrocytes in ( b , c ) using qPCR detecting gbb1 expression. Data are present as mean ± s.e.m. from three biologically independent experiments and analyzed with unpaired t test (two-tailed) to determine significance. *** P < 0.001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Staining, Transfection, Control, Shear, Expressing, Two Tailed Test

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 Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells were transfected with control siRNA and treated with shear stress, baclofen (100 μM) or ATP (100 μM) in the indicated time point. Data are present as mean ± s.e.m. from 15 cells recorded and are representative from six biologically independent experiments. b Percentage of astrocytes in response to shear stress or baclofen in 236 recorded cells. c Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells are transfected with siRNA knocking down the GABA B receptor and administered to shear stress, baclofen (100 μM), or ATP (100 μM) at the indicated time points. Data are present as mean ± s.e.m. from 45 cells recorded and are representative of six biologically independent experiments. d Percentage of astrocytes with the calcium response (sensitive) or without the calcium response (insensitive) to shear stress (control siRNA: 236 cells; GABA B siRNA: 314 cells). Data are analyzed with χ2 test (two-sided) to determine significance. **** P < 0.0001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Shear, Transfection, Control

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: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

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

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