Antibodies for β actin

Lipopolysaccharide (LPS, Salmonella enterica serotype enteritidis), pregnant mare serum gonadotropin (PMSG) and antibodies for β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid was purchased from Yakuri Pure Chemicals (Kyoto, Japan). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyCloneTM (Logan, UT, USA) and Penicillin-Streptomycin Solution was purchased from WELGENE Inc. (Daegu, Republic of Korea). Antibodies for interleukin-1β (IL-1 β) were purchased from R&D Systems (Minneapolis, MN, USA), and TNF-α was purchased from Cell Signaling Technology (Boston, MA, USA).

Cells and tissues were lysed in a buffer containing 10 mM Tris-HCl, pH 7.1, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride, supplemented with a protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) as previously described (Koo et al., 2012 (link)). The lysates were centrifuged at 10,000 g for 10 min to yield supernatants, which were stored at −70°C until use. Cell or tissue lysates were quantified, separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, IL, USA). For immunoblotting, immobilized proteins of interest were probed with primary antibodies and horse-radish peroxidase-linked secondary antibodies for chemiluminescence detection. Primary antibodies against p-PERK, COX-2, IκBα, PAI-1, and ATF6 were from Santa Cruz Biotechnology (CA, USA). Antibodies for ATF4, CHOP, and Flag were from Cell Signaling Technology (MA, USA). Antibodies for β-actin was from Sigma. Anti-GRP78 antibody was from Abcam (Cambridge, UK).