Radioimmunoprecipitation assay ripa buffer

A 2× qPCRBIO SyGreen 1-Step Lo-ROX kit was obtained from PCR Biosystems (Wayne, Pennsylvania, USA). We purchased the Bradford solution, blotting-grade blocker (nonfat dry milk), and polyvinylidene fluoride (PVDF) membrane from Bio-Rad (Benicia, CA, USA). All-trans retinoic acid, EA, GA, and LPS (from Escherichia coli O111 : B4) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific to β-actin (cat no. 4970S), TuJ1 (cat no. 5568S), ERK1/2, (cat no.4695S), pERK1/2 (cat no. 4376S), JNK (cat no. 9252S), or pJNK (cat no. 4671S) came from Cell Signaling Technology (Danvers, MA, USA). We obtained cytosine arabinoside hydrochloride, the IL-6 quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kit, radioimmunoprecipitation assay (RIPA) buffer, and XTT solution salt from Abcam (Cambridge, MA, USA). The TNF-α sandwich ELISA kit was purchased from BioLegend (San Diego, CA, USA). Ethanol and methanol were purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS), fungizone, minimal essential medium (MEM), penicillin, Roswell Park Memorial Institute (RPMI) medium 1640, and streptomycin were acquired from GIBCO (Waltham, MD, USA). The Griess reagent and tetramethylbenzidine (TMB)-stabilized substrate for horseradish peroxidase were purchased from Promega (Madison, WI, USA). Tri-RNA Reagent was purchased from Favorgen (Kaohsiung, Taiwan).

Protein samples were prepared by lysing cells with radioimmunoprecipitation assay (RIPA) buffer (Abcam) in the presence of sodium orthovanadate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride (PMSF), all purchased from Sigma. Protein concentration in the supernatants was done by ultracentrifugation using the Microcon^® Centrifugal Filter Devices (Merck Millipore). Protein concentration was determined with DC protein assay (Bio-Rad). Equal amounts of protein were separated with 10% Mini-PROTEAN TGX Gel (Bio-Rad) and then transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with HSP27, HSP70, HSP60, PD-L1, STAT3, phospho-STAT3 (Tyr705), beta- actin (Cell Signaling Technology) antibodies at 1:1,000 and NLRC5 rat mAb (Clone3H8, 1:1,000; Merck) overnight. Primary antibodies were detected with anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology) except for the NLRC5 rat monoclonal antibody which was detected with anti-rat IgG, HRP-linked antibody (Cell Signaling Technology) and developed with SignalFire^™ Enhanced Chemiluminescence (ECL) Reagent (Cell Signaling Technology).

TNBS, sodium thioglycolate, LPS from Escherichia coli O111:B4, and Roswell Park Memorial Institute (RPMI) medium were obtained from Sigma-Aldrich (USA). Radio immunoprecipitation assay (RIPA) buffer was obtained from Abcam (UK). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6, IL-1β, and TNF-α were purchased from eBioscience (USA). Antibodies against p-p65, p65, cyclooxygenase (COX)-2, inducible NO synthase (iNOS), and β-actin were procured from Cell Signaling Technology (USA) and Abcam (USA). A Pierce enhanced chemiluminescence (ECL) western blotting substrate was obtained from Thermo Fisher Scientific (USA). The Gram staining kit was obtained from BioMerieux (France).