Vinculin antibody

Proteins (15–20 µg) extracted in radioimmunoprecipitation assay buffer (50 mm Tris–HCl, pH 7.4, 1 mm ethylenediaminetetraacetic acid, 150 mm NaCl, 0.1% sodium dodecyl sulphate, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mm sodium fluoride, 1 mm orthovanadate) were separated using Bio-Rad 3–8% tris-acetate gradient gel (Bio-Rad) electrophoresis for Western blot analysis. Rabbit polyclonal BRCA2 (recognizes an epitope between residues 450-500) antibody (BETHYL lab, Cat # A303-434A-T-1, 1:2000 dilution), rabbit monoclonal GAPDH antibody (Cell Signaling technologies, Cat# 5174, 1:200,000 dilution) and mouse monoclonal Vinculin antibody (Santa Cruz biotech, Cat# sc25336, 1:200,000 dilution) were used to detect proteins. ECL plus Western blotting detection system (Amersham) was used for chemiluminescent detection. All Western blots were derived from the same experiment and were processed in parallel.

For Western blotting, cells were lysed in RIPA buffer (Santa Cruz Biotechnology, Dallas, TX) with protease and phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA). Protein concentrations were measured with Thermo Fisher Scientific Bradford Assay (Catalog No. PI23236). ETV5 antibody (Catalog No. ab102010) was purchased from Abcam, and CDK6 antibody (Catalog No. sc-7961) was obtained from Santa Cruz Biotechnology. GAPDH antibody (Sigma, G9545, Saint Louis, MO), ERK2 antibody (Santa Cruz Biotechnology, sc-1647), and vinculin antibody (Santa Cruz Biotechnology, sc-73614) were used as a loading control. Both goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). The fluorescent signals were captured with Odyssey CLX Imaging System (LI-COR Biosciences).

MEF cells (BALB/C-3T3) were plated on 6-well plates with a density 2 × 10⁵ cells/well in 2 ml medium and cultured for 48 h. Then cells were switched into serum-free medium and cultured for 24 h. Cells were next treated with 200 mIU/mL of Heparinase III (GlycoNovo Technologies, China) for 3 h. After that, 500 μg/mL of Heparin and 0.1–10 ng/mL of eGFP-bFGF were added into the medium respectively, incubated for 1 h. Cells were washed three times with PBS before collected and lysed with RIPA lysis buffer (Beyotime Biotechnology, China). The lysates were centrifuged for 10 min at 10,000x g and the supernatants were used for Western blot. Anti-ERK1/2 antibody (Santa Cruz Biotechnology, USA, 1:200 dilution) and anti-p-ERK 1/2 (pT202/pY204.22 A, Santa Cruz Biotechnology, USA, 1:100 dilution) as used for the detection of total ERK and phosphorylated ERK. Horseradish peroxidase–linked anti mouse IgG (Beyotime Biotechnology, China) was used as secondary antibody (1:2000 dilution). The signals were developed using BeyoECL Plus regent (Beyotime Biotechnology, China) and imaged with Chemiscope mini imaging system (CLINX, China). For protein loading control, vinculin antibody (Santa Cruz Biotechnology, USA, 1:200 dilution) was used. The results were analysed with ImageJ 1.53.