Highly pathogenic avian influenza A virus H5N1 non-structural protein 1 is associated with apoptotic activation of the intrinsic mitochondrial pathway

Bian, Qian; Lu, Jing; Zhang, Li; Chi, Ying; Li, Yan; Guo, Hongxiong

Western blot analysis. Monolayers of cells transfected with DNA or untransfected cells were lysed with ice-cold lysis buffer (150 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 tablet Complete Mini protein inhibitor mixture/10 ml buffer and 0.7 µg/ml pepstatin), and the total protein concentration was determined using the bicinchoninic acid assay. A total of 7 µl proteins with equivalent concentrations (1 µg/µl) were heated for 5 min at 100˚C in lysis buffer containing β-mercaptoethanol, and then resolved using 4-20% SDS-PAGE. The proteins were then transferred onto a polyvinylidene difluoride membrane and blocked with 1% powdered skimmed milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. Anti-cytochrome c MAb mouse antibody (1:200, BD556432; BD Biosciences) was then used to probe for cytochrome c overnight at 4˚C. The membrane was also probed for GADPH (1:1,000, A2066; Sigma-Aldrich; Merck KGaA) was used as the loading control. Membranes were subsequently washed with 150 mM PBS and incubated for 1 h at 4˚C in 10% dried milk in PBS. Membranes were washed 5 times with PBS and subsequently incubated for 1 h at room temperature with anti-mouse immunoglobulin G horseradish peroxidase-conjugated secondary antibody (1:1,000, ZB2307; ZSGB BIO; OriGene Technologies, Inc., Rockville, MD, USA)., followed by visualization of positive bands with the Pierce (Thermo Fisher Scientific, Inc.) enhanced chemiluminescence procedure using Kodak BioMax film. Blots were scanned and the protein ratios were calculated using the PDQuest program (version 7.4.0; Bio-Rad Laboratories, Inc.). The results shown are representative of 3independent experiments.