Hc101 01

Cells were cultured in 6-well dishes, harvested with EDTA-containing trypsin, centrifuged at 800× g for 3 min and washed twice with PBS. After discarding the supernatant, the cells were lysed in 40 μL of 1% NP40 containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail (EDTA-Free, MCE) for 30 min on ice. Subsequently, the insoluble fraction was removed by centrifugation at 15,000× g for 10 min at 4 °C, and the supernatant was transferred to a new tube with SDS sample buffer and boiled at 95 °C for 5 min. Samples were run on SDS-PAGE, followed by Western blotting analysis.
Rabbit monoclonal anti-BIP (C50B12, CST), rabbit monoclonal anti-PERK (D11A8, CST), rabbit monoclonal anti-IRE1α (14C10, CST), mouse monoclonal anti-ATF6 (66563-1-Ig, Proteintech), rabbit polyclonal anti-GRP94 (14700-1-AP, Proteintech), and mouse monoclonal anti-β-Tubulin (HC101-01, Transgene) were used as primary antibodies; goat anti-rabbit IgG, HRP (HS101, Transgene) and goat anti-mouse IgG, HRP (HS201, Transgene) were used as secondary antibodies. Core-fucosylated N-glycans were detected using Lens culinaris agglutinin (Bio-LCA, J-Chemical), followed by streptavidin-HRP (eBioScience). Thapsigargin (Tg) (100 nM; Sigma-Aldrich) was used for drug treatments.

Antibodies were as follows: mouse anti-NLRP3/NALP3 monoclonal antibody (AG-20B-0014, 1:2,000, Adipogen, San Diego, USA); rabbit anti-IL-1β monoclonal antibody (#12426, 1:2,000, Cell Signaling Technology, Beverly, MA, USA); rabbit anti-IL-18 polyclonal antibody (ab71495, 1:2,000, Abcam, Cambridge, UK); mouse anti-caspase-1 (p20) monoclonal antibody (AG-20B-0042, 1:2,000, Adipogen, San Diego, USA); and the appropriate IRDyeTM 800-conjugated secondary antibody (926-32210, 926-32211, 1:10,000, li-cor). Results were normalized relative to β-tubulin (HC101-01 1:5,000, Transgen Biotech, China) expression.