Anti mouse caspase 1 p10 antibody sc 514

Electrophoresis of proteins was performed using NuPAGE system (Invitrogen) according to manufacturer’s protocol. Briefly, BMDMs primed with LPS for 3 h were incubated in hypertonic conditions as described above for 3 h, lysed in lysis buffer [20 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 1 mM EDTA, and 1X protease inhibitor cocktails (Roche)] and stored at −20C until analyzed. For caspase-1 release, media supernatants were harvested after overnight incubation in hypertonic conditions as described above and stored at −20C until analyzed. Prior to electrophoresis, whole cell lysates or media supernatants were diluted with 5X protein sample buffer (125 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 0.06% bromophenol blue, and 1% β-mercaptoethanol) and incubated at 72°C for 10 min. Proteins were separated on 4%–12% NuPAGE gels and transferred to PVDF membrane (Millipore). To detect caspase-1, a rabbit polyclonal anti-mouse caspase-1 p10 antibody (sc-514; Santa Cruz Biotechnology) was used. LPS-primed BMDMs stimulated with ATP (5 mM) or Alum (250 μg/ml) for 30 min or 4 h respectively for caspase-1 activation in whole cell lysates, or for 2 h or 6 h respectively for caspase-1 release in media supernatants were used as positive controls.