anti β1 integrin subunit antibody  (R&D Systems)

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: Pull-down proteins were blotted with an anti-β1-integrin subunit antibody (#MAB1997, clone MB1.2, Merck-Millipore, Massachusetts).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: Subsequently, cells were divided into two equal parts and stained for 30 min with antibodies against total anti-β1-integrin subunit (10 μg/mL, #MAB1997, clone MB1.2, Merck-Millipore, Massachusetts) and against active anti-β1-integrin subunit (1:25, #MAB2247, clone 12G10, Merck-Millipore, Massachusetts).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: To pharmacologically block Rho/ROCK pathway and β1-integrin, hPAECs were pretreated with the ROCK inhibitor Y-27632 (#10005583, Cayman Chemical Company, Michigan) and an anti-β1-integrin subunit antibody (#MAB17781, clone P5D2, R&D Systems, Minnesota).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

doi: 10.1186/ar2024

Figure Lengend Snippet: Over-release of prostaglandin E2 (PGE 2 ) in compressed costal cartilage explants is the result of mechanical stress. (a) Implication of the mechanoreceptor integrin α5β1 in PGE 2 over-release in compressed cartilage explants. Mouse costal cartilage explants treated with either the β1 non-blocking antibody VMA1997 or the α5β1 blocking antibody AB1950 at 2.5 μg/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not-compressed control (cont) value. Data are the mean ± SEM of 2 independent experiments with n = 2/group/experiments, analyzed in duplicate. ***p < 0.001 versus control NC, *p < 0.05 versus control C. (b) Increased PGE 2 release in compressed costal cartilage explants is not due to the cytokine IL-1. Mouse costal cartilage explants treated with the IL-1 receptor antagonist (IL1-Ra) at 100 ng/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not compressed control value. Data are the mean ± SEM of 2 independent experiment with n = 2/group/experiments, analyzed in duplicate. *p < 0.05 versus control NC.

Article Snippet: Anti-goat fibronectin receptor (integrin α5β1) blocking polyclonal antibody (AB1950) was purchased from Euromedex for Chemicon Inc. (Strasbourg, France) and rat anti-mouse β1 subunit of VLA1 integrins non-blocking monoclonal antibody (VMA 1997), was purchased from AbCys SA for Chemicon Inc. (Souffelweyersheim, France).

Techniques: Blocking Assay