Horseradish peroxidase conjugated goat anti mouse or anti rabbit igg

Total protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH8.0, 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.0% NP-40) supplemented with a Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA). Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Protein (30-50 μg/lane) was separated by 10% SDS-PAGE and transferred on to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). After the blocking with 5% milk powder in TBS-T ( Tris-buffered saline - Tween20) for 1 hour, the membranes were incubated over night with primary antibodies including anti-β-catenin (9582, Cell Signaling Technology, Danvers, MA), anti-β-actin (A5441, Sigma), anti-IFIT1 (HPA055380, sigma), anti-IFIT2 (12604-1-AP, Proteintech Group, Chicago, MA), anti-PARP (9532, Cell Signaling Technology), anti-cleaved PARP (9541, Cell Signaling Technology), anti-Bax (sc-493, Santa Cruz Biotechnology, Dallas, TX), anti-Bak (sc-832, Santa Cruz Biotechnology), anti-caspase-3 (9662, Cell Signaling Technology), and anti-caspase-8 antibodies (M032-3, MBL, Nagoya, Japan). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (GE Healthcare) served as the secondary antibody for the ECL Detection System (GE Healthcare).

Lysates were extracted from the heads of E12.5 embryos, MEFs, and adult livers in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS) supplemented with a Protease Inhibitor Cocktail Set III (Calbiochem). Immunoblot analysis was performed using the following primary antibodies: mTOR (7C10, Cell Signaling), Notch1 (D1E11, Cell Signaling), NF-κB2 (18D10, Cell Signaling), Myc (Y69, Abcam), Klf5 (ab24311, Abcam and G-7, Santa Cruz), c-Jun (H-79 and G-4, Santa Cruz), TGIF (H-172, Santa Cruz), Cyclin E (C19, Delta Biolabs), SREBP1 (2A4, Novus Biologicals and H-160, Santa Cruz), and β-actin (AC-15, Sigma). Each primary antibody was used at a 1:1000 dilution. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (GE Healthcare) was used as the secondary antibody. Immunoreactive proteins were visualised using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions.