anti integrin α2 rabbit polyclonal antibody  (St Johns Laboratory)

Journal: International Journal of Implant Dentistry

Article Title: Effects of the application of low-temperature atmospheric plasma on titanium implants on wound healing in peri-implant connective tissue in rats

doi: 10.1186/s40729-024-00524-3

Figure Lengend Snippet: Connective tissue attachment. a Expression of adhesion-related genes during wound healing. Relative gene expression levels were quantified based on Day 3 of Cont. ** p < 0.01, * p < 0.05. b Immunohistochemistry of integrin α2, α5, and β1 during wound healing of peri-implant connective tissue. High-magnification histological evaluation during wound healing in peri-implant connective tissue is shown (bars: 20 μm). The regions of interest (ROIs) for integrin localization were set at the peri-implant connective tissue around the apical part of the peri-implant epithelium (shown with H&E staining, bar: 200 μm). Immunoreaction for each integrin was observed in the peri-implant connective tissue

Article Snippet: For primary antibody reaction, sections were incubated with the following primary antibodies for 2 h at room temperature: anti-integrin α2 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd., London, UK), anti-integrin α5 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd.), and anti-integrin β1 rabbit polyclonal antibody (1:200; Proteintech Group Inc., Chicago, IL, USA).

Techniques: Expressing, Gene Expression, Immunohistochemistry, Staining

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: Integrin α subunit cytoplasmic tails determine ECM-specific inflammatory signalling. (a) Alignment of integrin α subunit tail sequences and schematic representation of integrin α5/2 chimaera. (b,c) NF-κB activation by flow. WT α5 or α5/2 cells on FN (20 μg ml−1) were subjected to 30 min laminar shear (b) or 18 h oscillatory shear (c). NF-κB activation was then assayed by western blotting for Ser536 p65 phosphorylation (b, n = 3; c, n = 4). Y axis values throughout the figure represent the fold change (relative to control). LS, laminar shear; OS, oscillatory shear. (d) Induction of ICAM-1 after 18 h of oscillatory flow in cells expressing WT α5 versus the α5/2 chimaera on FN (n = 3). (e–g) NF-κB activation by soluble atherogenic factors. WT BAECs on FN or diluted Matrigel (MG) were stimulated with IL-1β or oxidized LDL for 30 min. NF-κB was assayed by western blotting for pSer536 p65 (e). t-p65, total p65. Quantification in f (n = 7) and g (n = 4) shows activation relative to control cells on FN without flow. (h) Effect of the chimaera on PKA activation. WT α5 or α5/2 chimaera cells on FN were sheared for 15 min and active PKA was pulled down from cell lysates with GST-PKI followed by immunoblotting. Forskolin (FSK) was used as a positive control for PKA activation (n= 4–8). (i) PKA inhibition rescues NF-κB activation in α5/2 chimaera cells. Chimaera cells on FN were treated with the PKI 14–22 amide inhibitor or dimethylsulfoxide (DMSO) alone, and then subjected to oscillatory shear for 18 h. Activation of NF-κB was assayed by western blotting (n= 4). Data are represented as means ± s.e.m. *P < 0.05 by one-way ANOVA (b,c,h,i) or two-tailed t-test (d,f,g). In all panels n values represent independent experiments. Source data are provided in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 6.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: Activation Assay, Western Blot, Expressing, Positive Control, Inhibition, Two Tailed Test

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: Integrin chimaera knock-in mouse showed reduced inflammation in artery. (a) Targeting strategy. These floxed, knock-in mice were then bred with CMV-Cre TG mice to obtain germline replacement of the exon containing the integrin α5 cytoplasmic domain with the integrin α2 cytoplasmic domain. (b) A genomic DNA fragment from homozygous knock-in mice containing the integrin α5 tail was amplified by PCR and sequenced. (c) Validation of integrin chimaera knock-in mouse. Lung homogenates from WT and chimaera knock-in mice were western blotted with the indicated antibodies. (d,e) Endothelial cells isolated from adult WT or chimaera knock-in homozygous mice were replated on dishes coated with poly-L-lysine or FN (10 μg ml−1). (d) After 15 min, adherent cells were quantified using the acid phosphatase assay and normalized to the cells attached on PLL (n = 3 independent experiments). Error bars are s.e.m. (e) Cell spreading at the indicated times was determined as described in Methods. For each condition, n= 20 images (~10 cells per field) were pooled across three independent experiments. The box plot shows the median, with upper and lower percentiles, and the bars show maxima and minima values. Source data for d are available in Supplementary Table 1. (f) Inflammatory markers in an athero-prone artery segment of knock-in mice. Aortae from WT and chimaera knock-in homozygous mice were stained for the indicated proteins and the lesser curvature of the arch was examined. Staining intensity was quantified as described in Methods (n = 5 mice for each type). L, lumen. Data are represented as means ± s.e.m. *P < 0.05 by two-tailed t-test. Quantification data from individual mice are provided in Supplementary Table 1.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: Knock-In, Amplification, Western Blot, Isolation, Acid Phosphatase Assay, Staining, Two Tailed Test

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: Involvement of PDE4D in ECM-dependent inflammatory signalling. (a) BAECs on FN were treated with the PDE4 inhibitor rolipram (1 μM), sheared, and PKA activity assayed as in Fig. 1. (n = 5). (b) BAECs on FN or collagen (Col.) were treated with rolipram (1 μM) and assayed for NF-κB as before (n = 3). (c) HUVEC lysates were immunoprecipitated with two different PDE4D5 antibodies and western blots probed for integrin α5 or integrin α2. Similar results were obtained in three experiments. (d) BAECs expressing WT integrin α5 or the α5/2 chimaera were immunoprecipitated with human-specific integrin α5 antibody recognizing the extracellular region. Western blots were probed for PDE4D5 and for the integrin β1 subunit. Similar results were obtained in 4 experiments. (e) BAECs were transfected with siRNA against PDE4D and then rescued with control or PDE4D5-GFP retrovirus. Cells were subjected to oscillatory shear and NF-κB assayed as before (n = 4). (f) BAECs expressing WT PDE4D5 or FAT-PDE4D5 were plated on Matrigel for 5 h and subjected to oscillatory shear. NF-κB Ser536 phosphorylation and Creb Ser133 phosphorylation were measured by western blotting (n= 4). Data are represented as means ± s.e.m. *P < 0.05 by one-way ANOVA (a,e,f) or two-tailed t-test (b). In all panels n values represent independent experiments. Source data are provided in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 6.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Expressing, Transfection, Two Tailed Test

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: Mapping the integrin binding site on PDE4D5. (a) Schematic representation of PDE4D5 and fragments used for pulldown assays. (b) HUVEC lysates were incubated with GST-tagged fragments of PDE4D5 and probed for integrin α5. Results are representative of three independent experiments. (c) To test whether the interaction is direct, integrin α tails immobilized on cobalt beads were incubated with purified PDE4D5 F2 fragment. Beads were washed and bound material was analysed by western blotting (α5R: scrambled sequence of α5 tail). (d) Deletion constructs used for detailed mapping and critical residues for binding. (e,f) The indicated PDE4D5 fragments and mutants were immobilized on GSH beads and incubated with the α5 tail protein used in c. Bound α5 tail protein was detected by western blotting with integrin α5 antibody against cytoplasmic tail. Results are representative of three independent experiments. (g) BAECs expressing GFP-tagged PDE4D5 WT or the 4E mutant were plated on FN or collagen and sheared for 15 min. The cells were fixed and stained for the focal adhesion marker, vinculin. Arrow indicates colocalization of PDE4D5 with vinculin. Results are representative of three independent experiments. Scale bar, 50 μm. (h) BAECs stably expressing integrin α5 binding-deficient PDE4D5 mutants (4A and 4E) or the catalytically inactive mutant (D556A) were transfected with siRNA to knock down the endogenous PDE4D, and then were subjected to oscillatory shear for 18 h. NF-κB activity was assayed as in Fig. 1; (n= 3 independent experiments). Data are represented as means ± s.e.m. *P < 0.05 by two-tailed t-test. Source data are provided in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 6.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: Binding Assay, Incubation, Purification, Western Blot, Sequencing, Construct, Expressing, Mutagenesis, Staining, Marker, Stable Transfection, Transfection, Activity Assay, Two Tailed Test

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: ECM-dependent regulation of PDE4D phosphorylation. (a) BAECs expressing WT or the 4E mutant of PDE4D5 were plated on collagen or FN for 5 h and then sheared for 15 min. Cell lysates were probed for anti-pSer651-PDE4D (n = 4–6). Y axis values throughout the figure represent the fold change (relative to control). t-PDE4D, total PDE4D. (b) BAECs expressing WT integrin α5 or the α5/2 chimaera on FN were transfected with PDE4D5, and then sheared for 15 min. Ser651 phosphorylation was assayed by western blotting as in a (n= 3). (c) BAECs expressing PDE4D5 WT or the 4E mutant or chimaera cells expressing PDE4D5 WT were kept in suspension (Sus.) for 90 min and then replated on FN-coated dishes for the indicated times. Ser651 phosphorylation was assayed by western blotting (n = 3). (d,e) BAECs in which endogenous PDE4D5 was knocked down were reconstituted with WT, phospho-deficient S651A or phospho-mimetic S651E mutants. The cells were replated on collagen (d) (n = 3) or FN (e) (n = 6) and then subjected to oscillatory shear for 2 h. NF-κB activity was assayed as in Fig. 1. In all panels n values represent independent experiments. Data are represented as means ± s.e.m. *P < 0.05 by one-way ANOVA (a,b,d) or two-tailed t-test (c,e). Source data are provided in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 6.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: Expressing, Mutagenesis, Transfection, Western Blot, Activity Assay, Two Tailed Test

Journal: Nature cell biology

Article Title: Interaction between integrin α 5 and PDE4D regulates endothelial inflammatory signalling

doi: 10.1038/ncb3405

Figure Lengend Snippet: In vivo PDE4D knockdown reduces flow-dependent inflammation. (a) Validation of siRNA used for in vivo knockdown. Immortalized mouse aortic endothelial cells were transfected with GFP-tagged human PDE4D5 and transfected with siRNA used for nanoparticle formulation. Endo., endogenous PDE4D5. NF-κB activation was assayed either by measuring GFP reporter expression under control of the NF-κB responsive element or ICAM-1 induction. Y axis values represent the fold change (relative to control). Data are represented as means ± s.e.m. *P < 0.05 and #P = 0.079 by two-tailed t-test. (n= 3 independent experiments) Source data are provided in Supplementary Table 1. (b) Nanoparticles containing PDE4D siRNA or luciferase (LUC) siRNA (1 mg kg−1) were injected intravenously. Aortae from treated mice were isolated and stained for the indicated molecules to assay inflammatory markers in lesser curvature (n= 5 mice). (c) Integrin chimaera knock-in mice were bred with ApoE null mice and fed a high-fat diet for 4 months. Aortae were opened and stained en face with Sudan IV (n= 4 mice). Plaque area and numbers were quantified. Data are represented as means ± s.e.m. *P < 0.05 by two-tailed t-test. Quantification data from individual mice are provided in Supplementary Table 1.

Article Snippet: Anti-p-NF-κB p65 (Ser536): rabbit monoclonal antibody (93H1), Cell Signaling (3033L), 1:1,000 for immunoblotting; anti-p-Creb (Ser133): rabbit monoclonal antibody (87G3), Cell Signaling (9198S), 1:1,000 for immunoblot-ting; anti-PKAc: mouse monoclonal antibody, BD Transduction Laboratories (610981), 1:1,000 for immunoblotting; anti-integrin α2: rabbit polyclonal, Millipore (AB1936), 1:1,000 for immunoblotting; anti-VCAM-1: rabbit monoclonal antibody (EPR5047), Abcam (ab134047), 1:200 for immunohistochemistry; anti-ICAM-1: rat monoclonal antibody (YN1/1.7.4.), BioLegend (116101), 1:200 for immunohistochemistry; anti-ICAM-1: rabbit polyclonal, Abcam (ab124759), 1:1,000 for immunoblotting; anti-NF-κB-p65: rabbit polyclonal (C-20), Santa Cruz (sc-372), 1:2,000 for immunoblotting; anti-vinculin: mouse monoclonal antibody (VIN-11-5), Sigma (V4505), 1:500 for immunohistochemistry; anti-fibronectin: rabbit polyclonal, Sigma (F3648), 1:400 for immunohistochemistry; anti-p-FAK (Tyr397): rabbit polyclonal, Cell Signaling (3283S), 1:1,000 for immunoblotting; anti-FAK: rabbit polyclonal, Cell Signaling (3285S), 1:1,000 for immunoblotting; anti-PP2A, C subunit: mouse monoclonal antibody (1D6), Millipore (05-421), 1:1,000 for immunoblotting.

Techniques: In Vivo, Transfection, Activation Assay, Expressing, Two Tailed Test, Luciferase, Injection, Isolation, Staining, Knock-In