Sc 30086

Western blotting was performed by following a routine protocol. Briefly, LCs were washed with phosphate buffered saline (PBS) and scraped into 200 μL sodium dodecyl sulphate (SDS) electrophoresis sample buffer. Cell lysates were then sonicated (60 Hz, 10 s for 3 times) and heated for 10 min at 95°C. Protein samples were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to a nitrocellulose membrane (NC membrane). The blotting membranes were blocked with 5% w/v non-fat milk and probed with rabbit anti-SF1 (1:800 dilution, Cell Signaling Technology, D1Z2A), rabbit anti-LRH1 (1:800 dilution, Abcam, ab189876), rabbit anti-CREB (1:1000 dilution, Cell Signaling Technology, 48H2), rabbit anti-CREM (1:1000 dilution, Abcam, ab64832) and rabbit anti-CYP19 (1:400 dilution, Santa Cruz Biotechnology, sc-30086) respectively. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000 dilution, Sigma-Aldrich, A0545) for 1 h at room temperature. The bound antibodies were visualized by a chemiluminescence system (BIO-RAD Biosciences). The optical densities of the bands were quantified by using Image J software (NIH). GAPDH served as loading control.