Orp150

Adult male SPF Sprague-Dawley rats, with weight from 200 g to 250 g, were provided by Hunan SJA Laboratory Animal Co. (Changsha, Hunan, China). All animals were housed under standardized environment with free access to standard laboratory rodent chow and sterile water. The study was approved by the Committee on Ethics of Animal Experiments of Wuhan University and performed in compliance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. 4-PBA and sodium taurocholate (STC) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Isoflurane was from Abbott Laboratories (Shanghai, China). EliVision super/horseradish peroxidase (HRP) kits were purchased from Maixin Biotech Co. (Fuzhou, China). Rat tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience Inc. (Vienna, Austria). Rat insulin ELISA kit was from Elabscience Biotechnology Co. (Wuhan, China). BiP, ORP150, CHOP, and Caspase-3 antibodies were purchased from Abcam (Cambridge, United Kingdom); insulin antibody was from Cell Signaling Technology Inc. (Danvers, MA, USA).

The following antibodies were used for WB and/or IF as indicated: SIL1 (Proteintech #24110-1-AP); ATF4 (Cell Signaling #11815); ATF6B (Proteintech #15794-1-AP); EIF2α (Santa Cruz #sc-11386); pho-EIF2α (Cell Signaling #3597s); PDI (Assay Design #SPA-891); BiP (BD #610979); ORP150 (Abcam #EP5891); LC3B (Cell Signaling #2775); NCL (Cell Signaling #87792S); NPM (Cell Signaling #3542S); CTHRC1 (Abcam #ab85739); Anti SQSTM1 (BD Bioscience #610833); LAMP1 (Abcam #ab25630); CNX (Cell Signaling #2679S); NES (Millipore #AB5922); CAV-1 (Cell Signaling #3238); and GAPDH (Santa Cruz #sc-32233). Hoechst #33342 from Life Technology was used to counterstain nuclei. For PCR amplification of the spliced and unspliced form of human XBP1, the following primers were used: FW 5′-AGCTCAGACTGCCAGAGATCG-3′; REV 5′-AATCCATGGGGAGATGTTCTA-3′ [35 (link)]. Powders, buffers, and other commonly used reagents were supplied by Sigma unless otherwise noted and were of analytical grade or higher.