Goat anti mouse il 1β antibody

Cell lysates were prepared from cultured BMDC (1 × 10⁶), BMM or HEK-293 cells, and from freshly isolated peritoneal macrophages (PM) (adherent peritoneal exudate population) or splenocytes (prepared by mechanical disaggregation and ammonium chloride lysis). Supernatants and lysates were diluted in sample buffer (BioRad) containing 1% 2-mercaptoethanol and heated at 80 °C for 5 min. Protein concentration was determined by modified Lowry (Bio-Rad, Berkeley, California, USA), and samples (20 μg protein) were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel. The primary antibodies were rabbit anti-mouse P2X₇R (Alomone, Jerusalem, Israel; 3 μg/ml), or goat anti-mouse IL-1β antibody (R&D Systems; Abingdon, UK; 0.1 μg/m) and the secondary antibodies were horse radish peroxidase-conjugated goat anti-rabbit IgG (AbD Serotec) or horse radish peroxidase-conjugated rabbit anti-goat IgG antibody (DAKO, Copenhagen, Denmark): 50 ng/ml or 0.25 μg/ml, respectively. A Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to visualize protein expression using hyperfilm (Amersham Biosciences/GE Healthcare, Little Chalfont, Buckinghamshire, UK) and a medical film processor (Xograph Imaging System, Compact).