Spa835

Cells were solubilized at 4°C in lysis buffer (50mM Tris [pH 7.5], 150mM NaCl, 1mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail for 30 min at 4°C followed by sonication of 10 short bursts of 2sec and supernatant recovered by centrifugation at 13,000 g for 30 min. Protein concentrations were determined, and equal amounts of proteins were separated on a 6% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane (PVDF). Membranes were blocked in 5% milk and immunoblotted with anti-hERG (1/1000 dilution, Alamone, Cat #APC-062) and anti-Hsp90 (1/1000 dilution, Stressgen # SPA835) antibody which were detected using HRP conjugated secondary antibodies followed by enhanced chemiluminescence (ECL) detection system (Bio-Rad). Tubulin (1/10,000 dilution, Sigma #T6199) was used as loading control Image densities were quantified using ImageJ (NIH) by integrating pixel densities of individual protein bands and normalized to tubulin

Antibodies for IRE1α (#3294), PDI (#3501), PERK (#3192), eIF2α (#5324) and its phosphorylated form at ser51 (#3398, p-eIF2α^ser51) GRP78 (used in the cancer experiment, #3177), P62 (#5114), Bcl-2 (#3498), p-ULK1^ser757 (#14202), ULK1 (#8054), p-JNK^{Thr183/Tyr185} (#4668), JNK (#9252), ATF4 (#11815), Beclin-1 (#3495), caspase 12 (#2202), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology. GAPDH (ab9485) antibody was purchased from Abcam (Cambridge, United Kingdom). LC3I and LC3II were measured by antibody (L7543) that was purchased from Sigma-Aldrich (St. Louis, MO, United States). Monoclonal primary antibodies were used for the detection of heat shock protein 70 (HSP70, StressGen, SPA-810), heat shock protein 60 (HSP60, StressGen, SPA-806), heat shock protein 90 (HSP90, StressGen, SPA-835), heat shock protein 47 (HSP47, StressGen, SPA-470) and thioredoxin interacting protein (TXNIP and MBL). Polyclonal primary antibodies were used to detect thioredoxin (TRX, IMCO, and ATRX-06), actin (Sigma, A-2066), heat shock protein 25 (HSP25, StressGen, and SPA-801), glucose-regulated protein 78 (GRP78, StressGen, SPA-826, used in the acute experiments). Horseradish peroxidase conjugated IgG secondary antibodies were used (Jackson ImmunoResearch Laboratories, PA, United States and StressGen and Zymed, San Francisco, CA, United States).