Perk 3192

Thapsigargin was purchased from Invitrogen. Tunicamycin was purchased from Enzo Life Sciences. 4-hydroxytamoxifen (4-HT) was purchased from Sigma; in all experiments involving 4-HT 0.1% ethanol, the diluent for 4-HT, was used and was without effect. Selumetinib (AZD6244/ARRY-142886) and AZD8055 were provided by Paul Smith, AstraZeneca, Alderley Park, Macclesfield, UK, whilst SCH772984 was purchased from Selleck Chemicals. ABT-263 was purchased from Santa Cruz Biotechnology. GSK2606414 and QVD-oPh were purchased from MERCK and Calbiochem respectively. Antibodies specific for BCL_XL (2762), BiP (3177), CHOP (2895), eIF2α (9722), p-eIF2α (Ser51) (9721), p-ERK1/2 (Thr202/Tyr204) (9106), IRE1α (3294), PARP (9542) and PERK (3192) were purchased from Cell Signaling Technology; ERK1 (610031) from BD Biosciences; BIM (AB17003) from Millipore; BMF (ALX-804-343) from Enzo Life Sciences; PUMA (3043) from ProSci; BAK (sc-832), BAX (sc-493), BCL2 (sc-7382) and MCL1 (sc-819) from Santa Cruz Biotechnology; Atg5 (A0731) and β-Actin (A544) from Sigma. Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad and the enhanced chemiluminescence (ECL) system from GE Healthcare was used for detection.

DMSO (472301), tunicamycin (T7765), thapsigargin (T9033), brefeldin A (B7651), actinomycin D (A1410), DRB (D1916) and Forskolin (F6886) were purchased from Sigma. Monosodium urate crystals were prepared from uric acid (U0881, Sigma) as previously described[30 (link)]. PolyI:C, flagellin, MDP and R837 were from Invivogen. All siRNA were purchased from Ambion. Different shRNA were cloned in pLKO.1 (Addgene #10878) and/or in Tet-pLKO-puro (Addgene #21915) vectors as previously described[31 (link)]. A complete list of siRNA and shRNA used is presented in S1 Table. IRE1α (#3294) and PERK (#3192) antibodies were from Cell Signaling Technology, ATF6 antibody (ab122897) was from Abcam, and XBP-1s antibody (clone 143F) was from Biolegend. ATF4 antibody for ChIP (sc-22800) was from Santa Cruz. NLRP1 antibody (AF6788) was from R&D Systems.

Total protein was extracted from U251 cells using RIPA buffer and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, protein samples were transferred to a polyvinylidene difluoride membrane. After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies at 4℃ overnight followed by secondary antibodies at room temperature for 1 h. The primary antibodies Bcl-2 (26593-1-AP), GRP78 (11587-1-AP), CHOP (15204-1-AP), and ATF6 (24169-1-AP) were purchased from PTG (Chicago, IL, USA). The primary antibodies pro-Caspase12 (35965), Cleaved-Caspase12 (35965), p-PERK (3179), and PERK (3192) were purchased from Cell Signaling Technology (Beverly, MA, USA). The specific bands were visualized using an enhanced chemiluminescence kit (Solarbio Science & Technology Co. Ltd., Beijing, China). ImageJ software was used for the quantification of target protein.