rabbit polyclonal anti integrin α5  (Cell Signaling Technology Inc)

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: Frontiers in Neurology

Article Title: SPECT Imaging of Acute Disc Herniation by Targeting Integrin α5β1 in Rat Models

doi: 10.3389/fneur.2022.782967

Figure Lengend Snippet: Representative SPECT images in the caudal spine at 1 day, 7 days, 1 month, and 2 months postinjury. (A) Radioactive signal accumulation was revealed in the punctured disc area of the injured disc at 1 day postinjury. The radioactivity peaked at 1 week and remained at a higher level until 2 months postinjury. A small amount of radioactivity was found in normal disks. (B) The radioactivity of the disc injury region in vivo was plotted against the post probe injection time. ** p < 0.01 compared to injured disks at 1 day, 1 month, and 2 months. (C) To validate the specificity of 99m Tc-3PisoDGR2 for integrin α5β1, we conducted 99m Tc-RGD imaging in a rat model of disc herniation. 99m Tc-RGD is the specific probe for integrin αvβ3, which was intravenously injected into the injury group ( n = 2) on day 7 postinjury. The experimental process was the same as before. Almost no focal signal was observed in the corresponding region relative to the normal disc of the RGD group.

Article Snippet: Sections were incubated with diluted rabbit polyclonal anti-integrin α5 antibody (1:100; Santa Cruz Biotechnology) or diluted rabbit monoclonal anti-integrin β1 antibody (1:100; Epitomics) at 4°C overnight.

Techniques: Single Photon Emission Computed Tomography, Radioactivity, In Vivo, Injection, Imaging

Journal: Frontiers in Neurology

Article Title: SPECT Imaging of Acute Disc Herniation by Targeting Integrin α5β1 in Rat Models

doi: 10.3389/fneur.2022.782967

Figure Lengend Snippet: Immunohistochemical staining of integrin α5 and integrin β1 in nucleus pulposus (NP) and annulus fibrosus (AF) cells. (A) The expression of integrin α5 was observed in normal AF and NP cells. The increased expression of integrin α5 appeared as early as 1 day postinjury, and the peak appeared at 7 days postinjury and remained visible until 60 days. (B) The expression of integrin α5 was observed in normal AF and NP cells. The increased expression of integrin β1 appeared as early as 1 day postinjury, and the peak appeared at 7 days postinjury and remained visible until 60 days. (C) Integrin α5- and β1-positive cells per unit tissue volume were plotted at various time points (1 day, 7 days, and 2 months) after rat disc puncture. ** p < 0.01 compared to normal disc and injured disc at 1 day and 2 months.

Article Snippet: Sections were incubated with diluted rabbit polyclonal anti-integrin α5 antibody (1:100; Santa Cruz Biotechnology) or diluted rabbit monoclonal anti-integrin β1 antibody (1:100; Epitomics) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B , targeting of TMEM2 to focal adhesions (FAs) does not require the cytoplasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 ( full length ) and mCherry-mTMEM2/Δcyto (Δ cyto ) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A , in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B , immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C – E , TMEM2 associates with integrins via extracellular interactions. C , cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3',3'-dithiobis(sulfosuccinimidyl propionate), and the lysates from these cells were immunoprecipitated with anti-mCherry antibody, followed by immunoblotting analysis with antibodies to α5, β1, and mCherry. D , the extracellular domain of TMEM2 directly binds α5β1. Binding between TMEM2 extracellular domain (ECD) and the α5β1 ECD heterodimer was analyzed by a pull-down assay. E , the ECD of TMEM2 directly binds αLβ2 (lymphocyte function-associated antigen-1). Binding between TMEM2 ECD and the αLβ2 ECD heterodimer was analyzed by a pull-down assay.

Article Snippet: After washing, precipitated materials were analyzed by SDS-PAGE and immunoblotting with mouse monoclonal anti-mCherry antibody (clone 8C5.5; horseradish peroxidase–conjugated, BioLegend; 677703), rabbit polyclonal anti-integrin α5 antibody (Proteintech; 10569-1-AP), and rabbit monoclonal anti-integrin β1 antibody (clone EP1041Y; Abcam; ab52971) with secondary horseradish peroxidase–conjugated anti-rabbit IgG (Bio-Rad Laboratories; 170-6515).

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane, Immunoprecipitation, Western Blot, Binding Assay, Pull Down Assay

Journal: The Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: A model for the role of transmembrane protein 2 (TMEM2) in integrin-mediated cell adhesion and migration. Our results suggest that TMEM2-dependent degradation of hyaluronan (HA) is critical for cells to form strong cell–matrix adhesion on HA-rich extracellular matrix (ECM). A , high levels of HA in the ECM are inhibitory to the direct engagement of integrins to their ECM ligands. B , in the presence of TMEM2, HA in the ECM is locally removed, which generates a microenvironment that is permissible to the direct integrin–ECM engagement. C , the association between TMEM2 and integrins promotes the FA formation and maturation via further removal of HA in the vicinity of the integrin–ECM engagement. D , this in turn facilitates integrin clustering, integrin-mediated downstream signaling, and cellular responses. See the text for further discussion.

Article Snippet: After washing, precipitated materials were analyzed by SDS-PAGE and immunoblotting with mouse monoclonal anti-mCherry antibody (clone 8C5.5; horseradish peroxidase–conjugated, BioLegend; 677703), rabbit polyclonal anti-integrin α5 antibody (Proteintech; 10569-1-AP), and rabbit monoclonal anti-integrin β1 antibody (clone EP1041Y; Abcam; ab52971) with secondary horseradish peroxidase–conjugated anti-rabbit IgG (Bio-Rad Laboratories; 170-6515).

Techniques: Migration

Journal: Scientific Reports

Article Title: Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration

doi: 10.1038/s41598-020-66375-1

Figure Lengend Snippet: Comparison of protein expression in cell sheets. ( a ) Cell sheets collected by the developed method (M d ) and conventional method (M c ) were lysed in SDS-PAGE sample buffer, and proteins were analysed by western blotting with the following antibodies: anti-fibronectin (220 kDa, first panel), anti-myosin heavy chain (MHC, 220 kDa, second panel), anti-leukaemia inhibitory factor receptor (LIFR, 190 kDa, third panel), anti-integrin α5 (150 kDa, fourth panel), anti-β-actin (45 kDa, fifth panel), anti-vascular endothelial growth factor (VEGF, 22 kDa, sixth panel), and anti-SDF-1 (10 kDa, seventh panel). Arrows indicate target bands. Relative protein quantities (Q) of ( b ) fibronectin, ( c ) MHC, ( d ) LIFR, ( e ) integrin α5, ( f ) VEGF, and ( g ) SDF-1 were measured using their band densities on western blots. Protein quantities were normalized to the band density of β-actin and expressed as the quantity relative to the conventional method (mean ± SD, n = 4). Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. .

Article Snippet: Then, the membranes were treated with the following antibodies in 5% Blocking One/Tris-buffered saline (TBS; pH 7.4) for 12 h with shaking: 0.4 μg/mL mouse anti-human fibronectin monoclonal antibody (ab194395; Abcam), 2 μg/mL mouse anti-chicken MHC monoclonal antibody (MF20; R&D systems, Minneapolis, MN, USA), 0.4 μg/mL rabbit anti-human LIFR polyclonal antibody (22779-1-AP; Proteintech, Rosemont, IL, USA), 500-fold dilution of rabbit anti-human integrin α5 polyclonal antibody (#98204; Cell Signaling Technology, Boston, MA, USA) 1,000-fold dilution of rabbit anti-mouse β-actin polyclonal antibody (#4967; Cell Signaling Technology), 1 μg/mL rabbit anti-human SDF-1 polyclonal antibody (41422; Signalway Antibody, College Park, MD, USA), and 0.5 μg/mL rabbit anti-human VEGF polyclonal antibody (R30265; NSJ Bioreagents, Carmel Mountain Ranch, CA, USA).

Techniques: Comparison, Expressing, SDS Page, Western Blot