Antibodies against nlrp3

IMP (purity >99%) was purchased from Yuanye (Shanghai, China). MIA and dimethyl sulfoxide were obtained from Sigma (St Louis, USA). Antibodies against NLRP3, ASC, Caspase-1, VEGF, TIMP1, TGF-β1 and HIF-1α were purchased from Abcam (Cambridge, UK). HRP-conjugated affinipure goat anti-rabbit IgG(H + L) (Proteintech Group, Inc., SA00001-2, 1:20,000), HRP-conjugated affinipure goat anti-mouse IgG(H + L) (Proteintech Group, Inc., SA00001-1, 1:20,000). ECL luminescent liquid (Shanghai Tianneng, 180–5001), protein marker (Shanghai Tianneng, 180–6003), BCA protein assay kit (Thermo Fisher, 23,227). Bovine serum albumin, fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), TRIzol and 0.25% trypsin-EDTA were purchased from Gibco (Life Technologies Corp., California, USA). TransStart Green qPCR SuperMix was obtained from Takara (Dalian, China). The primers and rat GAPDH Endogenous Reference were supplied by Sangon Biotech (Shanghai, China). Enzyme-linked immunosorbent assays (ELISA) kits for IL-1β and IL-18 were supplied by Invitrogen (Life Technologies Corp., California, USA). All other chemicals were of reagent grade.

Qingkailing (QKL) was purchased from Shenwei Pharmaceutical Co., Ltd. (No. Z13020935, China). The protease inhibitor, radioimmunoprecipitation assay (RIPA) lysis buffer, and enhanced chemiluminescence (ECL) reagent were obtained from Vazyme Biotech (Nanjing, China). The antibodies against GAPDH, p-AMPK, and AMPK were purchased from Cell Signaling Technology (Danvers, MA USA), and the antibodies against NLRP3, ASC, IL-1β and caspase-1 were obtained from Abcam (Cambridge, UK).

Cell samples were prepared in RIPA buffer or Laemmli buffer with inhibitors. For nuclear proteins, cell samples were collected and extracted following the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). Bis-tris or Tris-Gly gels were utilized to separate the protein samples after they were reduced and denatured. The proteins on the gels were transferred onto a PVDF membrane using a Bio-Rad transfer system. Antibodies against NLRP3 (1 : 1000) were purchased from Abcam (Cambridge, UK). Antibodies against GSDMD (1 : 100), IL-1β (1 : 100) and caspase-1 (1 : 100) were purchased from Santa Cruz (TX, USA). Antibodies against HIF-1α and caspase-1 (p20) were purchased from Novus Biologicals (CO, USA). Antibodies against total NF-κB P65 (1 : 1000) and phosphorylated NF-κB P65 (p-P65, 1 : 1000) were purchased from Cell Signaling (MA, USA). Antibody against actin (1 : 5000) was purchased from Absin (Shanghai, China). The secondary antibodies were purchased from Cell Signaling. The membranes were visualized using SuperSignal West Dura Substrate (Thermo Scientific, MA, USA), and the bands were detected with AI600 (GE Healthcare, IL, USA) and then quantified using the ImageJ program.