Rabbit anti p ire1α

Hepatic tissues and cells prepared with radioimmunoprecipitation (RIPA) buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1% NP40 and 0.1% SDS, supplemented with protease and phosphatase inhibitors (Millipore, USA). 50 μg lysates were loaded onto 10% SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). The proteins were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) according to the manufacturer’s protocol. Western blots were performed using antibodies against rabbit anti-p-AKT (Cell Signaling, #4060, 1:1000), rabbit anti-t-AKT (Cell Signaling, #9272, 1:2000), goat anti-TRB3 (Santa Cruz, sc-34211, 1:500), mouse anti-CHOP (Beyotime, #AC532, 1:1000), rabbit anti-BIP (Beyotime, #AB310, 1:1000), rabbit anti-p-eIF2α (Cell Signaling, #9721, 1:1000), rabbit anti-t-eIF2α (Cell Signaling, #9722, 1:1000), rabbit anti-p-IRE1α (Novus Biologicals, #NB100-2323, 1:1000), rabbit anti-t-IRE1α (Cell Signaling, #3294, 1:1000) and rabbit anti-GAPDH (Cell Signaling, #5174, 1:1000).

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).