Bradford analysis

To obtain protein lysates, frozen liver samples were homogenized in Tris buffer (100 mM Tris, 300 mM KCl, pH 7.6 with 0.1% Triton X-100 (Serva, Heidelberg, Germany)) using a TissueLyser (Qiagen) for 2 × 30 s at maximum speed. Cellular debris was removed by centrifugation (14,000× g, 15 min, 4 °C) and protein concentrations were determined by Bradford analysis (Biorad, München, Germany). After SDS polyacrylamide gel electrophoresis, gels were immunoblotted to nitrocellulose and blots were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 h. The following antibodies were used: Rabbit anti-Ferritin-H (151023, Abcam, Cambridge, UK; 1:500), rabbit anti-MT (192385, Abcam; 1:1000), rabbit anti-Ctr1 (129067, Abcam; 1:2000), rabbit anti-MTF-1 antibody (86380, Novus Biologicals, Centennials, US; 1:250), and rabbit anti-β-Actin (8227, Abcam; 1:10,000). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Chemicon, Hofheim, Germany; 1:50,000) served as secondary antibody. Intensities of identified bands were quantified densitometrically with the Luminescent Image Analyzer LAS-3000 system (Fujifilm, Tokyo, Japan). Protein expression was normalized to β-actin expression or Ponceau staining.

Protein content was determined by Bradford analysis (BIORAD) according to the manufacturer’s protocol. 15 µg protein sample was mixed 1:1 with native sample buffer (BIORAD) and loaded on a Mini-PROTEAN TGX Precast Gel (BIORAD). The PAGE was run at 4°C and low voltage for 2 hr in Tris/Glycine buffer (BIORAD). The gel was rinsed with H₂O for 5 min and stained with 50 ml staining solution (15 ml 3,3',5,5'-tetramethylbenzidine (TMBZ) solution (6.3 mM TMBZ in methanol) +35 ml 0.25 M sodium acetate solution (pH 5)) for 1 hr at room temperature (RT) while gently agitating. The gel was then incubated for 30 min at RT in the dark in presence of 30 mM H₂O₂. The background staining was removed by incubating the gel in a solution of isopropanol/0.25 M sodium acetate (3:7). Following scanning, the gel was completely destained in a solution of isopropanol/0.25 M sodium acetate (3:7) and stained with the BlueSafe stain (nzytech) for 10 min.

Cells were harvested by trypsinization. Samples were lysed in 50 mM Tris at pH 8.0, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, one tablet protease inhibitors= (EDTA-free; Roche) per 10 mL, and 30 μg/mL DNase I, (Sigma Aldrich). Protein concentration was measured by Bradford analysis (Bio-Rad, 500-0006). Total protein (80–100 µg) was resolved by SDS-PAGE. Proteins were electroblotted on Amersham Hybond-ECL nitrocellulose membranes (GE Healthcare) using a wet transfer system (Bio-Rad).

Cells were pretreated with the indicated concentrations of atorvastatin, simvastatin, or mevastatin for 24 h and stimulated with MSU crystals (0.3 mg/mL) for 24 h. Total proteins were lysed on ice for 15 min in a radio immunoprecipitation assay buffer (Thermo Scientific, Rockford, IL, USA) containing a protease inhibitor (Roche, Diagnostics, Mannheim, Germany). After centrifugation at 15,000 rpm for 10 min at 4 °C, the supernatants were equalized using the Bradford analysis (Bio-Rad, Hercules, CA, USA).
Cell lysates were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) by electrophoresis. The membranes were blocked in 5% skim milk (BD Bioscience, San Francisco, CA, USA) and probed with appropriate dilutions of primary antibodies. Antibodies to PPAR-γ and caspase-1 (Abcam, Cambridge, MA, USA), NLRP3 (Novus Biologicals, Littleton, CO, USA), IL-1β and anti-cleaved IL-1β (Cell Signaling, Danvers, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.
The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected using an ECL chemiluminescence kit (Thermo). Densitometry was analyzed and quantified using Quantity One software (Bio-Rad).

Cells were pelleted at 500g for 3 min and resuspended in RIPA buffer consisting of 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS and 140 mM NaCl, supplemented with protease inhibitors (Roche). Lysates were vortexed for 30 s and incubated on ice for 30 min. Lysates were spun at 20,000g for 10 min at 4 °C, and supernatants were quantified by Bradford analysis (Bio-Rad). Denatured proteins (10–20 μg) were loaded and run on polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% w/v milk in TBS-Tween (TBS-T, 0.1%). Primary and secondary antibody incubations were performed in 5% milk in TBS-Tween (0.1%) and membranes were washed with TBS-T, except for SCC2^NIPBL, which was incubated in 5% bovine serum albumin (Sigma) in TBS-T (0.1%). The signal was developed with Clarity Western ECL Substrate (Bio-Rad) or Immobilon western Chemiluminescent HRP substrate (Millipore) on the ChemiDoc Imaging System (Bio-Rad).