Anti nlrp3

Antibodies against rat phosphorylated AMP-activated protein kinase (phospho-AMPK) α and β, total AMPKα and AMPKβ, phosphorylated protein kinase A catalyst unit (phospho-PKA C), sirtuin-1, caspase 3 (CASP3), and GAPDH were purchased from Cell Signaling (Danvers, Mass). Anti-GRP78/BIP and anti–peroxisome proliferator-activated receptor α (PPAR-α) antibodies were purchased from Abcam (Cambridge, Mass). Anti-NLRP3 and anti–PGC-1α antibodies were purchased from EMD Millipore (Billerica, Mass). SuperSignal West Pico chemiluminescent substrate was purchased from Thermo Scientific Inc. (Rockford, Ill).
Approximately 40 mg of frozen liver tissue was homogenized in 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), 1% (wt/vol) NP-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 1 mmol/L β-glycerol phosphate, 2.5 mmol/L sodium pyrophosphate, and 1× complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, Ind). The homogenate was centrifuged at 12,000g for 30 min at 4°C, and the pellet discarded. Western blotting was performed with 30 µg of protein per well. Band intensities were quantified with the Image J software (National Institutes of Health, Bethesda, Md). GAPDH was used as loading control.

1 × 10⁶ of virus-induced DCs were washed with chilled PBS and sonicated in 55 µl of 2× SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue). The extracts were heated at 95°C for 5 min. Equal quantities of solubilized protein were resolved by 10% SDS-PAGE, blotted to nitrocellulose membrane and probed with following antibodies: anti-NLRP3 (EMD Millipore),, anti-ASC (Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain), anti-caspase-1, anti-cleaved caspase-1 (Cell Signaling) and anti-β-actin antibody (Sigma-Aldrich). Proteins were visualized with SuperSignal West Pico chemiluminescent substrate (Pierce). For co-immunoprecipitation, cells were harvested in RIPA buffer (Sigma Aldrich), and cell lysates were incubated with anti-caspase-1 overnight at 4°C followed by incubation with protein A Dynabeads (Invitrogen) for 2 h. The beads were washed three times with PBS, suspended in Laemmli buffer (62.5mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenol blue), boiled for 10 min and spin down. Supernatants were collected and analyzed for the expression of NLRP3 and ASC by immunoblotting.