Horseradish peroxidase conjugated goat anti mouse igg antibody

The tissues and HUVECs were homogenized with modified RIPA buffer and stationed on ice for 1 h, following which they were centrifuged at 14,000 × g at 4°C for 5 min. The supernatants were collected, mixed with loading buffer containing 0.1% bromophenol blue and boiled for 10 min, following determination of protein concentration by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Equal quantities of protein (10 µg) were loaded into a 10% SDS-PAGE gel, followed by electrophoresis, separation under denaturing conditions and electroblotting onto PVDF membranes. The membranes were incubated overnight in Tris-buffered saline containing 7% milk to inhibit nonspecific antibody binding. The proteins of interest were revealed via incubation with specific mouse anti-human monoclonal antibody (anti-FLIP_S/L; 1:500 dilution; cat. no. sc-5276; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature, followed by incubation with a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (cat. no. A0216; Beyotime Insititute of Biotechnology) for 1 h at room temperature. Signals were visualized using chemiluminescence. β-actin antibody was used as a control. All western blots were quantified using densitometry.