Horseradish peroxidase hrp conjugated rabbit anti mouse secondary antibody

The cells were harvested and lysed in a cell lysis buffer including protease inhibitor cocktail (Sigma). Extracts were separated by centrifugation at 14,000g for 15 min at 4 °C. SDS-PAGE and Western blot analysis were performed. Briefly, equal amount of proteins were boiled in 1× SDS sample buffer and run in a 10% SDS-PAGE gel, and transferred onto pure nitrocellulose blotting membranes (Pall Corporation). The membrane was blocked with 3% nonfat dry milk solution in PBS at room temperature for 2 h and rinsed three times with PBS before incubating with primary antibodies. After that, the immunoblots were probed with anti-biotin (1:500 dilution; Santa Cruz Biotechnology), anti-Trx1 (1:500 dilution; Santa Cruz Biotechnology), anti-PC (1:500 dilution; Santa Cruz Biotechnology) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary antibody (1:10,000 dilution; Sigma). Anti-GAPDH primary antibody (1:10,000 dilution; Sigma) and HRP-conjugated rabbit anti-mouse secondary antibody (1:5,000 dilution; Sigma) were employed to normalize protein expression after the membranes were stripped by incubating in a buffer containing 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mM Tris–HCl (pH 6.8). The blots were developed using chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Biosciences).