Ecl advance system

The kidney tissues were homogenized and the supernatant was collected after centrifugation at 12,000 ×g at 4°C for 20 min [23 (link)]. We separated the lysates on 10% polyacrylamide gels before immunoblotting using anti-Nlrp3 (AdipoGen company, San Diego, CA), anti-ASC (AdipoGen company, San Diego, CA), anti-CHOP (Cell Signaling Technology, USA), anti-caspase-12 (Cell Signaling Technology, USA), anti-IL-1β (Affinity Biosciences, USA), and anti-IL-18 (Affinity Biosciences, USA) antibodies at a dilution of 1 : 500. The expression levels of CHOP, caspase-12, ASC, Nlrp3, IL-1β, and IL-18 were analyzed using an ECL advance system (Amersham, Little Chalfont, UK). The relative protein expression levels were determined by normalization to β-actin. Serum IL-1β and IL-18 were measured with ELISA kits (RayBiotech, Norcross, GA) according to the manufacturer's instruction.