Protease inhibitor cocktail

Liver tissues were washed with ice-cold PBS and the protein extracts were prepared using ice-cold cell lyses buffer supplemented with protease inhibitor cocktail (IBI SCIENTIFIC, Peosta, USA). Protein concentrations were measured using Bradford assay (Bio-Rad, CA, USA) according to the manufacturer's protocol. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.25% Tween 20, pH 7.5) at room temperature for 2 h followed by incubation with 2 μg/mL of primary antibody for GPx (sc-22145), CAT (sc-34285), GST (ab19256), PON-1 (sc-59646), PON-3 (sc-21156), PPAR-δ (ab8937), ABCA-1 (sc-58219), MCP-1 (sc-28879), and GAPDH (sc-32757) diluted in TBS and 5% skimmed milk overnight at 4°C. After washing with TBS-T buffer, the membrane was incubated with 1 μg/mL of horseradish peroxidase (HRP) labeled secondary antibody diluted in TBS-T buffer for 2 h at room temperature, followed by three washes with TBS-T buffer. The detection was performed using chemiluminescence assay (Amersham, GE Healthcare, Life Sciences, UK). Membranes were exposed to X-ray film to observe the bands (Kodak, Rochester, NY). Protein bands in treated and untreated (control) groups were quantified using the Kodak Scientific ID software.