Anti smac

Protein was isolated from homogenised samples and quantified using the BCA kit (Sigma-Aldrich, Huangpu, Shanghai, China). Ten percent SDS PAGE was done to separate the proteins and then transferred to a nitrocellulose membrane by means of electroblotting technique. Following this, the membrane was blocked using 5% blocking solution (non-fat milk). The membrane was kept in the blocking buffer containing primary antibodies overnight at 4°C (rabbit anti-cytochrome c 1 : 500, anti-smac 1 : 1000, anti-HIF-1α 1 : 500, anti-p53 1 : 1000, anti-FASL 1 : 300, anti-caspase-8 1 : 1000, anti-BID 1 : 500 and anti-β-actin 1 : 1000 (Santa Cruz Biotechnology, Chai Wan, Hong Kong, China). Next day, goat secondary antibody conjugated with horseradish peroxidase was added in the dilution 1 : 1000 in blocking buffer (non-fat milk). A chemiluminescence kit (Thermo Fisher Scientific, Pudong, Shanghai, China) was used to detect the proteins. The sample used was the mitochondrial fraction isolated from the rat spinal cord, as previously described [20 (link)].