Phospho eif2α ser51

Curcumin, AO, MDC, NAC, z-VAD, CHX, and DCF-DA were purchased from Sigma (St. Louis, MO, USA). The class III PI3K inhibitor 3-MA was obtained from Calbiochem (La Jolla, CA, USA). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI, USA). Antibodies against PARP, caspase-3, p62, Beclin-1, CHOP, phospho-eIF2α (Ser51), IRE1-α, Bip/GRP78 and Nrf2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against LC3 (Sigma) were also used. The anti-β-actin, anti-ubiquitin, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

For protein extraction, tissue was homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktail (#A32959, Thermo Scientific) and protein concentration was measured using the DC™ Protein Assay Reagent (#500‐0114, BioRad). The following primary antibodies were used: phospho‐AMPK (Thr172) (#2531, Cell Signaling Technology), total AMPK (#2603, Cell Signaling Technology), phospho‐eIF2α (Ser51) (#3597, Cell Signaling Technology), eIF2α antibodies (#3524, Cell Signaling Technology), OXPHOS antibody (#ab110413, Abcam), slow myosin (#ab11083, Abcam), and UCP1 antibodies (#ab23841, Abcam). Protein expression was normalized to α‐Tubulin (ATUB) (#T6074, SIGMA). Horseradish peroxidase‐conjugated secondary antibodies were used: anti‐rabbit IgG (#7074, Cell Signaling Technology) or anti‐mouse IgG (#7076, Cell Signaling Technology).

B cells or LPS-stimulated plasmablasts were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitors (Roche) and phosphatase inhibitors. Protein concentrations were determined using BCA assays (Pierce). Protein samples were boiled in SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromophenol blue) with β-ME, analyzed by SDS-PAGE, and transferred to nitrocellulose membranes, which were subsequently blocked in 5% nonfat milk (wt/vol in PBS), and immunoblotted with indicated primary antibodies and appropriate horseradish-peroxidase-conjugated secondary antibodies (Southern Biotech). Primary antibodies to IRE-1 (Cell Signaling Technology), XBP-1 (Cell Signaling Technology), PERK (Santa Cruz), phospho-eIF2α (Ser51; Cell Signaling Technology), eIF2α (Cell Signaling Technology), ATF4 (Cell Signaling Technology), GRP94/BiP (anti-KDEL; Enzo Life Sciences), p97 (Fitzgerald), actin (Sigma-Aldrich), μ heavy chain (Southern Biotech), and κ light chian (Southern Biotech) were obtained commercially. Polyclonal antibodies against mouse class I MHC heavy chain, class II MHC α, β, or invariant (li) chains, Igβ, and STING were generated in rabbits. Immunoblots were developed using Western Lightning Chemiluminescence Reagent (PERKinElmer).

b-AP15 was obtained from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO at a stock concentration of 10 mM, aliquoted and stored at −80°C. Other agents are N-acetyl-L-cysteine (NAC, Sigma-AldrichInc., St. Louis, MO); pan caspase Inhibitor Z-VAD-FMK (EnzoLife Sciences International, Inc, Plymouth Meeting, PA). Antibodies (Abs) used in this study were purchased from following sources: anti-CDK2, CDK4, CDK6, anti-cyclinD1, anti-p27, anti-PARP, anti-Bcl-2 (50E3), anti-Bim (Y36), anti-Noxa (D8L7U), anti-BIP (C50B12), anti-CHOP (L63F7), eIF2α, phospho-eIF2α (Ser51), anti-HSP70, anti-HSP90, anti-AIF, cytochrome C and anti-Phospho-MDM2 (Cell Signaling Technology, Beverly, MA, USA); anti-Rb, phospho-Rb (S780), anti-cleaved caspase-8 (Cleaved Asp384) (Assay biotechnology Company, Inc); anti-cleaved caspase-9 p35 (D315), anti-cleaved caspase-3 (p17), anti-AR, anti-MDM2 and anti-GAPDH (Bioworld Technology, Inc). anti-p53(Abcam), anti-ubiquitin (P4D1), anti-K48-linked tetra-ubiquitin, Bax (B-9) (Santa Cruz Biotechnology, Santa Cruz, CA); MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). CCK-8 assay kit, PI and Annexin V-FITC apoptosis Detection Kit, DCFH-DA and cell apoptosis Rhodamine 123 Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies.

Colonic tissues were homogenized in a radioimmunoprecipitation assay buffer (50 mM Tris-Cl [pH 8.0], 320 mM sucrose, 0.1 mM EDTA, 1 mM DTT, 1% Nonidet P-40, 0.1% SDS and 1% protease/phosphatase I and II inhibitor cocktail [Sigma]). Proteins (50–100 µg) were separated on 10% SDS-PAGE gel, transferred onto a membrane using iBlotGel Transfer device (Invitrogen), and probed with primary antibodies: Phospho-eIF2α (Ser⁵¹) (Cell Signalling), eIF2α, ATF4, and GADD34 (Santa Cruz Biotechnology), KDEL (Abcam), and β-actin (Sigma). Horseradish peroxidase-conjugated secondary antibodies were detected using ECL reagents (Pierce).

Antibodies used in the study were: HSPA5 (Santa Cruz Biotechnology, sc‐376768); EIF2S1 (Santa Cruz Biotechnology, sc‐133132); ATF4 (Cell Signaling Technology, 11815s); Phospho‐eIF2α (Ser51) (Cell Signaling Technology, 3597s); EIF2A (Proteintech, 11233‐1‐AP); LC3 (Proteintech, 12135‐1‐AP). Paclitaxel and Adriamycin were purchased from Sigma.