Iκb antibody

Cell extracts were prepared by lysing cells cultured in 6-well plates with RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% NaDOAc, 0.1% SDS, and 1.0%NP-40) plus phosphatase and protease inhibitor cocktails (Xpert P3200-001 and P3100-001, respectively, GenDEPOT). Protein concentrations were determined by the Bio-Rad method (DC Protein Assay), and equal amounts of proteins were subjected to SDS-PAGE gels (8–15%). Proteins were transferred onto PVDF membranes and incubated with primary antibody overnight at 4 °C, followed by a 1 h incubation with secondary goat anti-mouse or goat anti-rabbit Alexa-Fluor 680 (Molecular Probes), or donkey anti-goat 780 (LI-COR Biosciences). Primary antibodies include NLRP3 antibody (Adipogen), phospho-p65 antibody, IκB antibody, total p65 antibody and phospho-p38 antibody (Cell Signaling Technology), total p38 antibody and β-actin antibody (Santa Cruz Biotechnology). The results were visualized using Li-Cor Odyssey Infrared Imaging System (LI-COR Biosciences).

DMEM, fetal bovine serum (FBS) (Gibco/invitrogen, Carlsbad, CA, USA), a Cell Counting Kit-8 assay (Dojindo Lab, Kumamoto, Japan), β-glucan kit (Megazyme International, Wicklow, Ireland), Phagocytosis Assay Kit (Cayman, MI, USA), cyclophosphamide (Sigma Aldrich, St.Louis, MO, USA) β-1,3-glucan (Sigma Aldrich, St.Louis, MO, USA) and cordycepin (Sigma Aldrich, St.Louis, MO, USA) were purchased. Phospho-Lyn antibody, Lyn antibody, phospho-Syk antibody, Syk antibody, phospho-ERK antibody, ERK antibody, phospho-p38 antibody, p38 antibody, phospho-JNK antibody, JNK antibody, NFκB antibody, phospho-IκB antibody and IκB antibody were obtained from Cell signaling Technology Inc. (Danvers, MA, USA). β-Actin antibody was obtained from Santa Cruz (Dallas, TX, USA).