Pro parp

ZIKV-E antibody (#B1845) was purchased from Beijing Biodragon Immunotech (Beijing, China). Antibodies for pro-caspase-1 antibody (#ab179515), phospho-MLKL (#ab187091), phospho-RIPK3 (#209384) and GSDMD (#ab210070) were purchased from Abcam (Cambridge, MA). Another GSDMD antibody (#A10164) was purchased from ABclonal. Antibodies for NLRP3 (#D4D8T), pro-caspase-3 (#9555S), cleaved caspase-3 (#9664S, #9661), pro-PARP (#9532S), cleaved-PARP (#9541S), and phospho-RIPK1 (#44590S) were purchased from Cell Signalling Technology (Beverly, MA). We also purchased antibodies for cleaved caspase-1 (#AF4022) from Affinity Biosciences (Taizhou, China) and GAPDH and β-actin from Proteintech (Wuhan, China). An MTT Assay Kit was purchased from SunShineBio (Nanjing, China). ELISA kits for mouse interleukin-1β, mouse interleukin-18, human interleukin-1β, and human interleukin-18 were purchased from BOSTER (Wuhan, China). Compounds VX765, ZVAD-FMK, GSK’872, Nec-1s, and Phorbol-12-myristate-13-acetate (PMA, an activator of protein kinase C for activating monocytes) were purchased from Selleck. RNAiso plus reagent was purchased from TAKARA.

The protein was isolated from cells with lysis buffer (pH=7.4, 1% NP-40, 1 mM Na₃VO₄, 1 M EDTA, 1 mM NaF, 50 mM Tris-Hcl, 0.25% sodium deoxycholic acid, 150 mM NaCl) containing protease inhibitor cocktail (Amresco, Scolon, OH, USA). Protein quantification was normalized with β-actin using a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA). The differences of protein expression were determined by Western blotting using SDS-PAGE 8% and 10% gel by electrophoresis. After blocking in 3% skim milk, the membrane with protein was probed with various primary antibodies for p-AKT, pro-PARP, CHOP (Cell signaling, Beverly, MA, USA), PI3K, and β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) for 24 h followed by exposure to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies for 1 h. Protein expression levels were identified by the chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ, USA).

The harvested cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA), and the lysates were centrifuged at 14,000× g for 20 min. The supernatants were removed, and the concentration of proteins measured at 280 nm, using the Bradford solution (Bio-Rad, Hercules, CA, USA) and a microplate reader (Molecular Devices). A 20 mg aliquot of lysate from each well was loaded onto an SDS-PAGE gel, electrophoresed, and transferred to a nitrocellulose membrane. After blocking for 1 h with 5% skim milk, the membranes were incubated at 4 °C with primary antibodies in 5% BSA solution at 4 °C, followed by washing and incubation with secondary antibodies for 1 h at room temperature. Bands were detected with Clarity^TM Western ECL Substrate (Bio-Rad) and visualized with Amersham ImageQuant 800 (GE Healthcare Bio-Sciences Corp., Marlborough, MA, USA). The primary antibodies used in this study included antibodies to pro-caspase-3, pro-caspase-9, cleaved caspase-3, cleaved caspase-9, pro-PARP, and cleaved PARP (Cell Signaling Technology, Beverly, MA, USA). An anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.