Pierce crosslink magnetic ip kit

The following primary antibodies were used for Western blot, co-IP, and immunofluorescence: anti-HAX-1 antibody (BD, cat: 610825), anti-NLRP3 antibody (CST, cat: 15101), anti-ASC antibody (CST, cat: 67824), anti-Caspase1 antibody (CST, cat: 24232), anti-Cleaved Caspase1 antibody (CST, cat: 89332), anti-GSDMD antibody (CST, cat:93709), anti-TMEM119 antibody (Abcam, cat: ab209064), anti-β-actin antibody (Abmart, cat: P30002S), Goat anti-rabbit antibody HRP linked (CST, cat: 7074), Goat anti-mouse antibody (CST, cat:7076). The co-IP assays were performed by using Pierce Crosslink Magnetic IP Kit (Thermo Scientific, Cat: 88805). The immunofluorescence assays were performed by using Opal 7-color manual IHC kit (PerkinElmer, NEL801001KT). The following recombinant proteins were used for BLI assay: recombinant NLRP3 (Abmart, cat: EHH22785), recombinant ASC (Proteintech, cat: Ag28424), recombinant HAX-1 (Proteintech, cat: Ag27244). NLRP3 selective inhibitor MCC950 was purchased from APExBIO company, cat: C3780.

Whole-cell lysates or cell nuclear lysates were prepared from human mandible-derived BM-MSCs. Immunoprecipitation experiments were extracted by using Pierce™ Crosslink Magnetic IP Kit (Thermo Scientific, Waltham, MA, USA) and a Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific, Waltham, MA, USA). An immunoprecipitation assay was performed as recommended by the supplier. Proteins extracted from 2×10⁶ human mandible-derived BM-MSCs was mixed with 1 μg of antibody and prewashed Protein A/G, then incubated overnight. The bound antigens were eluted from the beads by boiling samples for 10 min. Eluted samples were obtained from SDS-PAGE. Immunoblotting was carried out as previously described 36 (link). Proteins were extracted from mandibles as previously 13 (link). Primary antibodies against Sirt1, FOXO3a, acetylated-lysine, Histone H3, and β-actin (Cell Signaling Technology, Danvers, MA, USA) were used. The immunoreactive bands were visualized by ECL chemiluminescence (Amersham) and analyzed by the Scion image Beta 4.02 (Scion, National Institutes of Health).