B 0311p hrp

Bidicinchoninic acid (BCA) kit provided by Beyotime Biotechnology (Shanghai, China) was adopted to assay the protein that was isolated from LC cells. Using SDS-PAGE (10%), the protein was then extracted and converted onto PVDF membranes provided by Millipore (Billerica, MA, USA). Next, primary antibodies were applied to the membranes overnight at 4°C using 5% skimmed milk. The antibodies are as follows: anti-Bax (1: 2, 000, bs20337R, Bioss, Woburn, MA, USA), anti-Bcl-2 (1: 2, 000, bs20352R, Bioss, Woburn, MA, USA), anti-Cleaved caspase-3 (1: 2, 000, bs33199M, Bioss, Woburn, MA, USA), anti-MMP-2 (1: 2, 000, bs20705R, Bioss, Woburn, MA, USA), anti-MMP-9 (1: 2, 000, bs7095R, Bioss, Woburn, MA, USA), anti-PYCR2 (1: 2, 000, bs19690R, Bioss, Woburn, MA, USA) and anti-β-actin (1: 2, 000, bs0061R, Bioss, Woburn, MA, USA), with β-actin which was treated as the endogenous control. In the next step, membranes were further incubated with a secondary antibody (1: 2,000, b-0311P-HRP, Bioss, Woburn, MA, USA) for an additional hour. Subsequently, protein blots were viewed using ECL (Millipore, Billerica, MA, USA).