Rabbit mabs

TRIzol^TM (catalog #15596018) was purchased from Life Technologies (Carlsbad, CA, USA). A High-Capacity cDNA reverse transcription kit (catalog #4368813) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). A qPCR^TM core kit for SYBR Green I (catalog #10-SN10-05) was purchased from Eurogentec (San Diego, CA, USA). 2-NBD glucose (catalog #7133) was purchased from Setareh Biotech (Eugene, OR, USA). Tunicamycin (catalog #T7765), oleate (catalog #O1008), palmitate (catalog #P0500) and sulfo-N-succinimidyl oleate sodium (catalog #SML2148) were purchased from Sigma Aldrich (St. Louis, MO, USA). PERK (catalog #5683), IRE1α (catalog #3294), ATF6 (catalog #65880), BIP (catalog #3177), CHOP (catalog #5554), pAS160 (catalog #8730), pAKT (catalog #4060), PERK (catalog #45899) and ACTIN (catalog #4970) Rabbit mABs were purchased Cell Signaling (Danvers, MA, USA). HRP anti-rabbit secondary IgG antibodies (catalog #ab288151) were purchased from Abcam (Cambridge, MA, USA). All other chemicals and solvents were obtained from Fisher Scientific through its local distributor in the Kingdom of Saudi Arabia.

Cell lysates were obtained from primed THP-1 cells or BMDM after crystal stimulation. Cells were washed with ice-cold PBS and lysed for 30 min on ice with Triton X-100 based-buffer plus phosphatase (orthovanadate, Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were collected after centrifugation (7,500 g, 15 min) and total protein concentration was determined with the BCA Pierce Assay (Thermo Fisher Scientific). An amount of 30 μg protein was used for SDS-PAGE separation and transferred to PVDF membranes for western blot analysis. Anti-phosphorylated (P-) or total p38, p42/44 (ERK1/2) and JNK rabbit mAbs (1/1,000), as well anti-NLRP3 (1/500) rabbit mAbs were purchased from Cell Signaling. Anti-tubulin pAb was from Santa Cruz Biotechnology (1/1,000), anti-actin pAb was from Sigma-Aldrich (1/5,000).