Enhanced chemiluminescence detection kit for hrp

Cells were washed three times with pre-colded PBS solution and whole cell proteins were extracted by incubation with the radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China). The nuclear extracts were prepared using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA) according to the manufacturer’s instructions. Both protease and phosphatase inhibitor (Roche, Switzerland) were contained in the protein extracts. Protein concentration was determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein (50 μg) were separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and electrophoretically transferred to poly-vinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Immunoblots were visualized by Enhanced Chemiluminescence Detection Kit for HRP (Biological Industries, Israel) using Kodak X-OMAT LS film (Eastman Kodak, USA). The blots were subsequently stripped through incubation in TBST buffer and re-probed for GAPDH, lamin B1, or β-actin as a loading control. Quantitative data were obtained using Quantity One Software (Bio-Rad).

Tissues and cells were lysed in lysis buffer, and whole proteins were extracted by incubation with the western blot assay buffer (Beyotime, Nantong, China). Protein concentration was measured using a BCA protein assay kit (Beyotime, Nantong, China). A total of 50 µg of each protein sample was subjected to 10% SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature, and then incubated with a 1:1,200 dilution of anti-maspin monoclonal antibody (BD Pharmingen, San Diego, CA, USA) overnight at 4°C. The HRP-conjugated secondary antibody was incubated for 1 h at 37°C. Specific bands were visualized using the enhanced chemiluminescence detection kit for HRP (Biological Industries, Kibbutz Beit Haemek, Israel) and Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA). Quantitative data were obtained using Quantity One software.