Beyoecl plus regent

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.