Enhanced chemiluminescence assay kit

Cells were treated with ST (62.5, 125, 250 and 500 µg/ml) for 5 days and then washed twice with ice-cold PBS. The cell lysates were prepared with radioimmunoprecipitation cell lysis buffer (GenDEPOT, Barker, TX, USA). The lysates were centrifuged at 15,928 × g for 15 min at 4°C. The concentration of protein was measured using the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 30 µg of each protein was separated by electrophoresis using 4–20% Criterion™ TGX™ precast gels (Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). The membranes were blocked with 5% skim milk and incubated with primary antibodies (1:1,000 dilutions; β-actin (cat. no. sc-81178), PPAR-γ (cat. no. sc-7273), C/EBPα (cat. no. sc-61); Santa Cruz Biotechnology, Inc., Dallas, Texas. USA) overnight at 4°C. The next day, the membranes were incubated with goat anti-rabbit secondary antibodies (1:5,000; cat. no. 170-6515; Bio-Rad Laboratories, Inc.) for 1 h at room temperature, and immunoreactive proteins were detected using an enhanced chemiluminescence assay kit (Thermo Fisher Scientific, Inc.). Bands were detected using a ChemiDoc™ XRS + image analyzer (Bio-Rad Laboratories, Inc.).

The cells were lysed using RIPA buffer (Thermo Fisher Scientific), and the protein concentrations were determined using a BCA assay kit (Thermo Fisher Scientific). Equal amounts of proteins were obtained after SDS-PAGE separation and analyzed using primary antibodies against cFos, ERK, p-ERK, and p-IκBα (Cell Signaling Technology), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). The bands were visualized using an enhanced chemiluminescence assay kit (Thermo Fisher Scientific) and luminescent image analyzer (LAS-3000 system; Fujifilm, Tokyo, Japan). For quantification, ImageJ software (National Institutes of Health) were used to analyzed the bands intensity.