anti integrin α 3 β 1  (Bioss)

Journal: Nature medicine

Article Title: A tripartite complex of suPAR, APOL1 risk variants and α v β 3 integrin on podocytes mediates chronic kidney disease

doi: 10.1038/nm.4362

Figure Lengend Snippet: APOL1 forms high-affinity interactions with suPAR and αvβ3 integrin. All sensorgrams were generated using SPR assays. Rate constants (ka and kd) were determined by kinetic fitting of the sensorgrams using a one-to-one binding equation, and equilibrium dissociation constant (KD) values were determined by calculating kd/ka. (a) Sensorgrams of suPAR (0–300 nM) binding to immobilized APOL1 protein variants (G0, G1 and G2), and calculated KD values. (b) Sensorgrams of αvβ3 integrin (0–10 nM) binding to immobilized suPAR in the absence (inactive form) and presence (active form) of Mn2+ (0.5 mM), and calculated KD values. (c,d) Steady-state affinity fitting curves of αvβ3 (c) or α3β1 (d) integrin (0–40 nM) binding to immobilized APOL1 (G0, G1 and G2) in the absence (inactive) and presence (active) of Mn2+ (0.5 mM). (e) Sensorgrams of αvβ3 integrin (0–20 nM) binding to immobilized APOL1 (G0, G1 and G2) in the presence of Mn2+ (0.5 mM), and calculated KD values. (f) Bar graphs showing the determined KD values for suPAR in the presence of αvβ3 (5 nM), increasing concentrations of suPAR (0–300 nM) and immobilized APOL1 proteins (G0, G1 and G2) in the absence (inactive) or presence (active) of Mn2+ (0.5 mM). The average KD values were determined from two independent experiments, and error bars for KD measurements indicate s.e.m. (n = 2) (a–f). A, analyte; IM, immobilization.

Article Snippet: Human recombinant protein integrin α v β 3 (Millipore, cc1018, R&D, 3050-AV), human integrin α 3 β 1 (R&D, 2840-A3), human HDL full-length protein (Abcam, ab77897) and human suPAR (R&D, 807-UK-100/CF) were purchased.

Techniques: Generated, Binding Assay

Journal: Nature medicine

Article Title: A tripartite complex of suPAR, APOL1 risk variants and α v β 3 integrin on podocytes mediates chronic kidney disease

doi: 10.1038/nm.4362

Figure Lengend Snippet: High levels of suPAR are needed to synergize with APOL1 G1 and G2 to induce αvβ3 integrin activation on human podocytes. (a,b) Representative western blot images for immunoprecipitation assay using HEK293T cells transfected with plasmids expressing APOL1 G0, Flag-tagged suPAR and Myc-tagged β3 integrin in different combinations, as indicated. Data are representative of at least three independent experiments. The empty pFlag-CMV3 vector was used as a negative control. (a) Cell lysates were immunoprecipitated using a mouse monoclonal anti-Flag (M2) or monoclonal anti-Myc antibody. (b) Cell lysates were immunoprecipitated using a rabbit polyclonal anti-APOL1. The cell lysates and immunoprecipitates were analyzed by immunoblotting with antibodies, as indicated. Analysis of the input (1%) indicates that the levels of APOL1, β3 integrin and suPAR are overexpressed in the transfected cells. IP, immunoprecipitation. (c) Bar graph representing level of activated β3 integrin detected in human podocytes cultured in healthy serum without (Con) or with added proteins: suPAR (2,000, 5,000 and 10,000 pg/ml), APOL1 G0, G1 and G2 (15 µg/ml) or a combination of both proteins. Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). ***P < 0.001. Statistical significance was determined by unpaired two-tailed Student’s t-test. (d) Immunofluorescence analysis of human podocytes grown in human serum in the absence (Con) or presence of suPAR (5,000 pg/ml) and three APOL1 variant proteins (15 µg/ml). Cells were stained with AP5 antibody that specifically recognizes the active form of β3 integrin, and with anti-paxillin antibody to mark focal adhesions. Scale bar, 25 µm. (e) Bar graph representing the level of β3-integrin activation detected in human podocytes cultured in healthy serum without (Con) or with three serum samples from individuals with FSGS carrying the nonrisk APOL1 genotype, and three different serum samples from individuals with FSGS carrying two APOL1 risk-allele genotypes. FSGS-APOL1 serum #2, and to a substantial degree, serum #3, detached the cells such that the quantification of β3-integrin activation was either not determined (n.d.) or not accurate (striped bar graph). Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test. (f,g) Bar graph representing the level of β3-integrin activation detected in human podocytes cultured in healthy serum without (Con) or with the addition of the indicated concentrations of the proteins. In g, the concentration of suPAR was 5,000 pg/ml. Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). *P < 0.01; ***P < 0.001. Statistical significance was determined by unpaired two-tailed Student’s t-test.

Article Snippet: Human recombinant protein integrin α v β 3 (Millipore, cc1018, R&D, 3050-AV), human integrin α 3 β 1 (R&D, 2840-A3), human HDL full-length protein (Abcam, ab77897) and human suPAR (R&D, 807-UK-100/CF) were purchased.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Negative Control, Cell Culture, Two Tailed Test, Immunofluorescence, Variant Assay, Staining, Concentration Assay

Journal: Contrast Media & Molecular Imaging

Article Title: Synthesis and Bioevaluation of Iodine-131 Directly Labeled Cyclic RGD-PEGylated Gold Nanorods for Tumor-Targeted Imaging

doi: 10.1155/2017/6081724

Figure Lengend Snippet: (a) Immunofluorescence staining of integrin α v β 3 in B16F10 (upper row) and MCF-7 (lower row) cells. The nucleus were counterstained with DAPI. The red fluorescence intensity is proportional to the expression level of integrin α v β 3 (×200). (b) Results of cell binding assays at various time points.

Article Snippet: The expression of integrin α v β 3 was confirmed by immunofluorescence with a primary anti-integrin α v β 3 antibody (1 : 100, Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit secondary antibody (1 : 50, Aspen, Wuhan, China) as described previously [ ].

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Binding Assay

Journal: Contrast Media & Molecular Imaging

Article Title: Synthesis and Bioevaluation of Iodine-131 Directly Labeled Cyclic RGD-PEGylated Gold Nanorods for Tumor-Targeted Imaging

doi: 10.1155/2017/6081724

Figure Lengend Snippet: (a) Autoradiography of tumor tissue and organs. B16F10 tumor-bearing mice (upper row) and MCF-7 tumor-bearing mice (lower row). (b) Immunofluorescence staining of integrin α v β 3 in B16F10 (upper row) and MCF-7 (lower row) tumors (×200).

Article Snippet: The expression of integrin α v β 3 was confirmed by immunofluorescence with a primary anti-integrin α v β 3 antibody (1 : 100, Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit secondary antibody (1 : 50, Aspen, Wuhan, China) as described previously [ ].

Techniques: Autoradiography, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet–Mediated Amyloid Aggregation in Alzheimer’s Disease

doi: 10.3390/ijms22179633

Figure Lengend Snippet: Impact of complex III inhibition on platelet functions following Aβ40 stimulation. ( A ) Detection of intracellular ATP levels after incubation of platelets with Aβ40 (5 µM), antimycin A (12.5 µM) and EtOH (as control, vehicle) for 90 min using Luminescence intensity (n = 4, *** = p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). ( B ) Measurement of ATP release from antimycin A-pretreated platelets (EtOH was used in controls as vehicle) following Aβ40 (20 µM) or CRP (1 µg/mL) (n = 5–6). Analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons post-hoc test. ** p < 0.01; n.s.: not significant. ( C ) Aggregation of antimycin A-pretreated platelets upon Aβ40 (10 µM) or CRP (1µg/mL). EtOH was used as control (vehicle) (n = 5–6). ( D ) Measurement of reactive oxygen species (ROS) generation with DCF-DA in GPVI-deficient platelets upon Aβ40 and CRP (n = 6). Data represent mean value ± SEM; two-way ANOVA with Sidak’s multiple comparisons test. *** p < 0.001; ** p < 0.01; * p < 0.05. ( E ) Flow cytometric analysis of integrin activation at the surface of platelets using PAC-1 antibody upon stimulation with Aβ40 (11.5 µM) and ADP (5µM). Where indicated, samples were pre-incubated with vitamin C (1 mM) for 30 min at RT (n = 4). Data represent mean value ± SEM; two-way ANOVA with Tukey’s multiple comparisons test. *** p < 0.001; ** p < 0.002.

Article Snippet: Flow cytometry analysis of platelet activation was performed using fluorophore-labeled antibody PAC-1 (activated integrin α IIb β 3 receptor marker, BD Biosciences).

Techniques: Inhibition, Incubation, Activation Assay

Journal: Bone Research

Article Title: Ciliary parathyroid hormone signaling activates transforming growth factor-β to maintain intervertebral disc homeostasis during aging

doi: 10.1038/s41413-018-0022-y

Figure Lengend Snippet: PTH induces integrin α v β 6 expression to activate latent TGF-β. a Immunostaining images showing various types of integrin expressions in IVD tissue from 18-month-ld mice injected with PTH or vehicle and quantitative analysis ( b ). Scale bar, 50 μm. c qRT-PCR analysis of the mRNA levels of various integrin in NP tissue from 18-month-old mice injected with PTH or vehicle. Results reported as fold change. d Western blot analysis of integrin β 6 expression in NP cells of 18-month-old mice at different time points post PTH injection (PTH1-34, 100 nmol·L -1 ). e , f Chromatin immunoprecipitation assay with four different potential pCREB binding sites (primers 1, 2, 3 and 4) in the β6 integrin promoter. g pCREB, Integrin α V β 6 , pSmad2/3, or Safranin-O staining of IVD sections from an IVD ex vivo compression model of 30-month-old rat with treatment of either vehicle or PTH (PTH1-34, 100 nmol·L -1 ). Scale bar, 20 μm. h Quantitative analysis of the percentage of pCREB, pSmad2/3 positive cells and the Integrin α V β 6 positive areas as a percentage of total IVD area (Ar) of ( g ). All data are reported as the mean ± s.d. * P < 0.05. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t -test

Article Snippet: Sections for immunostaining were processed using a standard protocol and incubated with primary antibodies to rabbit ACAN (Abcam, 1:100), CCN2 (Abcam, 1:100), integrin β 8 (Abcam, 1:200), pCREB (Abcam, 1:100), and PTH1R PRB-635P (Covance, 1:100), mouse pSmad2/3 (Santa Cruz, 1:100), integrin α v β 6 (Millipore, 1:100), integrin α v β 3 (Bioss, 1:100), integrin α v β 5 (Bioss, 1:100) at 4 °C overnight.

Techniques: Expressing, Immunostaining, Injection, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Staining, Ex Vivo

Journal: Experimental eye research

Article Title: Normalization of Wound Healing and Stem Cell Marker Patterns in Organ-Cultured Human Diabetic Corneas by Gene Therapy of Limbal Cells

doi: 10.1016/j.exer.2014.10.022

Figure Lengend Snippet: Antibodies Used in the Study

Article Snippet: Although there was some typical staining variability among individual cases, possibly due to differences in donor ages and/or disease duration and severity, the presented pictures are representative of most studied cases. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Antigen Antibody Source Dilution MMP-10 Goat pAb sc-9941 Santa Cruz Biotechnology (Dallas, TX) 1:10 Cathepsin F Goat pAb AF2075 R&D Systems (Minneapolis, MN) 1:25 of 0.5 mg/ml stock c-Met Goat pAb sc-161 Santa Cruz Biotechnology 1:10 Keratin 14 Mouse mAb NB100-65109 Novus Biologicals (Littleton, CO) Undiluted Keratin 15 Mouse mAb sc-47697 Santa Cruz Biotechnology 1:10 Keratin 17 Mouse mAb sc-58726 Santa Cruz Biotechnology Undiluted ANp63 Goat pAb sc-8609 Santa Cruz Biotechnology 1:200 Fibronectin Mouse mAb 618 Ugarova et al. 1996 Undiluted Laminin γ3 chain Rabbit pAb R96 Saghizadeh et al. 2011 1:500 Nidogen-1 Mouse mAb MAB2570 R&D Systems 1:50 Integrin α 3 β 1 Mouse mAb MAB1992 EMD Millipore (Billerica, MA) 1:50 p-EGFR (Tyr845) Rabbit pAb 44-784G Thermo Fisher Scientific (Waltham, MA) 1:50 p-Akt (Ser473) Rabbit pAb 9271 Cell Signaling Technology (Danvers, MA) 1:50 p-p38 (Thr180/Tyr182) Rabbit pAb 05-1059 EMD Millipore Undiluted Open in a separate window mAb, monoclonal antibody; pAb, polyclonal antibody.

Techniques: