Chemiluminescence assay

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.

Cells were lysed in protein lysis buffer (Beyotime). The whole cell lysis products were analyzed with SDS-PAGE and then transferred to PVDF membrane (Millipore). The following primary antibodies were used: anti-KLF4 polyclonal antibody (ab106629, Abcam), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), and anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech). After incubation with the corresponding antibodies, the membrane was washed 3 times for 5 min each with TBST. We used ImageJ software for further densitometric analysis. Bound primary antibodies were detected by incubating for 1 h with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit IgG (Biofly, China) for analysis. The membrane was washed and developed by a chemiluminescence assay (GE Healthcare).

To prepare whole cell lysates, cells were collected by centrifugation and washed once with PBS and then lysed in EBC lysis buffer (Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8856) [50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40] supplemented with aprotinin (11.5 μg/ml), leupeptin (11.5 μg/ml) phenylmethylsulfonyl fluoride (50 μg/ml), NaF (100 mM) and Na ortovanadate (0.2 mM). Protein concentration was determined by BCA following the manufacturer's instructions (G Biosciences). Proteins (25 μg) were resolved in SDS/PAGE and transferred to PVDF filters. Blots were incubated with a mouse antibody against p73 (ER-15) (Thermo-Pierce) and then incubated with an anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz). Bound antibody was detected by a chemiluminescence assay (GE Healthcare).