Rabbit anti human bip

Total cell protein was extracted from cells using ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.0% Triton X-100, 1 tablet/10 cc buffer of PhosSTOP [Roche Applied Science, Mannheim, Germany], and protease inhibitor cocktail) and protein concentration was quantified using the Bradford method [20 (link)]. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then blotting was performed as described previously [13 (link), 18 (link)]. The primary antibodies included mouse anti-human REIC/Dkk-3 (Momotaro-Gene Inc., Okayama, Japan), rabbit anti-pIRE1α (Novus Biologicals, Littleton, CO, USA), rabbit anti-human BiP, rabbit anti-human β-catenin, TBP (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-human β-actin antibody (Sigma, St. Louis, MO, USA); all primary antibodies were diluted 1:2000 in Can Get Signal® (Toyobo Co., Ltd., Osaka, Japan). The secondary antibody horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology) was diluted 1:5000 in 1% skim milk [7 (link)]. We quantified band densities using Image J (ver,1.53r).

A rabbit anti-human IFNAR1 (Abcam Inc., Cambridge, MA), a mouse anti-human IL-10Rβ (IFNλR, R&D Systems Inc., Minneapolis, MN), rabbit anti-human BiP, IRE1α, peIF2α, CHOP, Beclin 1, ATG5, LC3B, and GAPDH (Cell signaling Technology, Beverly, MA), IFNAR2, IFNγR1, CNT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), HCV-core (Thermo Fisher Scientific, Waltham, MA), HCV-NS3 (Virogen, Watertown, MA), ENT1 (Abgent, San Diego, CA). A horseradish peroxidase-coupled antirabbit or antimouse IgG was used as secondary antibodies obtained from Cell Signaling Technology, Beverly, MA. Control siRNA (siIRR) and siRNAs against PERK and ATG7 (Life Technologies, Carlsbad, CA) were as described previously [18] (link).