Cw0098a

Whole-cell lysates were prepared using RIPA buffer. Equal amounts of total protein (30 μg) from cell lysates were loaded on a 6% SDS/PAGE gel, transferred to a PVDF membrane (Millipore), and detected using an ECL Western Blotting Detection System (Biorad). Primary antibodies were primary antibodies against ATRX (ab97508; Abcam; 1:1000), β-tublin (CW0098A; CWBIO; 1:5000). Secondary antibodies used were goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (Zhongshan Gold Bridge Biotechnology). Immunoblots were quantified using Image Lab 5.1 software.

LPS (L2630) and ATP (A7699) were purchased from Sigma-Aldrich. Nigericin (HY-127019), JANEX-1 (HY-15508), and A419259 (HY-15764) were from MedChemExpress (Monmouth, NJ, USA). Vigofect (T001) was from Vigorous (Beijing, China). The Lipofectamine™ RNAiMAX transfection reagent was from ThermoFisher (Waltham, MA, USA). The anti-rabbit ASC antibody (1:1000; #67824) was from Cell Signaling Technology (Cambridge, MA, USA). The anti-mouse NLRP3 (1:1,000; AG-20B-0014) and anti-mouse caspase 1 (p20) (1:1,000; AG-20B-0042) antibodies were from AdipoGen (San Diego, CA, USA). The anti-mouse IL-1β antibody (1:1,000; AF-401-NA) was from R&D Systems (Minneapolis, MN, USA). The anti-rabbit HCK antibody (1:500; A14537) was from Abclonal (Wuhan, China) and anti-rabbit HCK antibody (1:200; 11600-1-AP) was from ProteinTech (Wuhan, China). Anti-mouse β-tubulin (1:2,000; CW0098A), anti-mouse GAPDH (1:3000; CW0100M), goat-anti mouse horse radish peroxidase (HRP) IgG(H + L) (1:5,000; CW0110S), and goat anti-rabbit HRP IgG (H + L) (1:5,000; CW0103S) were from CWBiotech (Beijing, China). The anti-mouse FLAG antibody (1:1,000; F1804) was from Sigma-Aldrich and anti-mouse MYC antibody (M047-3) was from MBL (Woburn, MA, USA). Goat anti-mouse IgG HRP (L) and goat anti-mouse HRP(Fc) were from Invitrogen.

For immunoblotting, we used the following affinity-purified antibodies: anti-Smad2/3 (1:1,000; 3102, Cell Signaling Technology), anti-p-Smad2 (1:500; AB3849, Millipore), anti-Smad3 (1:1,000; 9523, Cell Signaling Technology), anti-p-Smad3 (1:1,000; 04-1042, Millipore), anti-Fscn1 (1:5,000; 5535-1, Epitomics), anti-FoxA2 (1:1,000; 8186S, Cell Signaling Technology), anti-Nanog (1:1,000; Ab80892, Abcam), anti-Sox17 (1:1,000; 09038, Millipore), anti-Actin: (1:1,000; CW0096A, CWBIO), anti-Tubulin (1:1,000; CW0098A, CWBIO), anti-Myc (1:3,000; M20002, Abmart), anti-HA (1:3,000; M20003, Abmart) and anti-Flag (1:5,000; F3165, Sigma). Anti-Myc (1:100) and anti-HA (1:100) antibodies were also used in immunoprecipitation assays.
For each immunoprecipitation assay, either HEK293T or HEK293 cells were transfected with indicated plasmids. Cells were collected 48 h after transfection and lysed with TNE lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail. Lysates were incubated with protein A sepharose beads and indicated antibodies at 4 °C for 4 h. The beads were then washed four times with TNE buffer. The bound proteins were separated by SDS–PAGE and visualized by western blotting. The intensity of each band was quantified using Image J. Full images of uncropped western blots are shown in Supplementary Fig. 10.