Antibodies against bax and bcl 2

Cells were lysed in urea lysis buffer (7 M urea, 0.1 M DTT, 0.05% Triton X-100, 25 mM NaCl, 20 mM Hepes pH 7.5). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (SDS PAGE), transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridised to an appropriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. Primary antibody against β-actin was purchased from Abcam (Cambridge, UK). Antibodies against Bax and Bcl2 were purchased from Santa Cruz Biotechnology (CA, USA). The secondary antibodies, HRP-conjugated rabbit anti-mouse IgG and swine anti-rabbit IgG, were obtained from DakoCytomation (Glostrup, Denmark).

Nova was prepared according to method described previously [9 ], in brief, a shuffling library with 12 subtypes of IFNαs was constructed from human leukocyte cDNAs. Followed by PCR amplification, DNase I digestion and anti-tumor/virus activity screening, Novaferon, a human interferon-like protein was screened. The purity of Nova was determined by Size Exclusion Chromatography-High- Performance Liquid Chromatography (SEC-HPLC) assay. Nova was separated by Shodex Protein KW-803 column (Showa Denko America, Inc., NY, USA) in PB buffer (20 mM Na3PO4, 0.5 mM NaCl, 0.02% Tween80, pH7.0). The purity was calculated by division of the target peak area and the total peak area.
Antibody against caspase-3 cleaved fragment was purchased from Upstate Biotechnology (Temecula, CA, USA). Antibodies against Bax and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against Ki-67 was purchased from Thermo Fisher Scientific (Melbourne, VIC, Australia). Antibody against α-SMA was purchased from Abcam (Cambridge, MA, USA). Recombinant human IFN-α/β R2-Fc chimera, recombinant human IgG1 Fc and goat anti-human IgG Fc was purchased from R&D Systems (Minneapolis, MN, USA). FITC Annexin V/PI apoptosis kit was from BD Biosciences (Burlington, MA, USA).

3-(4,5-Dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and MG (40%, w/v), Propidium Iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luteolin (purity > 98%) was purchased from Shanxi Sciphar Biotechnology Co., Ltd (Shanxi, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). mTOR, phospho-mTOR (Ser2448), phospho-4E-BP1 (Thr37/46), AKT, phospho-AKT (Ser473), phospho-p70S6K (Thr389) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Cytochrome C antibody was purchased from Abcam (Cambridge, UK). Antibodies against Bax and Bcl-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-β-actin monoclonal antibody was purchased from Abmart (Shanghai, China). All secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).