Anti star

Anti-StAR (Abcam, Cambridge, UK) and actin (Sigma) antibodies were commercially available. Whole-cell extracts were resolved on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The blots were blocked in Tris-buffered saline (10 mM Tris, pH 7.6, 150 mM NaCl, and 2 mM MgCl₂) containing 0.3% Tween 20 and 3% bovine serum albumin and incubated with primary antibody at room temperature for 1 hour. Antibody binding was detected by incubation with secondary antibodies linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) accompanied by visualization using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, MA). Optical densities of immunoreactive bands were quantified using NIH ImageJ software (downloaded from http://rsb.info.nih.gov/ij/), and the relative amounts of target proteins were deduced by comparison with optical band densities from serially diluted reference extracts.

For western blot analysis, cells were harvest in a 10 mM Tris (pH 7.4) buffer containing 1% SDS and 1 mM Na3VO4 on ice and 20 µg of cell lysate were separated by 10% SDS-PAGE and electro-transferred onto a nitrocellulose membrane as described elsewhere. The blots were then washed in Tris-buffered saline (20 mM Tris-HCl, pH 7.6 containing 137 mM NaCl and 0.05z (vWv) Tween 20), blocked with 5% skim milk for 1 h, incubated with primary antibody (1:1000) at 4°C for overnight. Primary antibodies used for immunoblotting were anti-actin (Santa Cruz, USA; #sc-47778), anti-CYP11A1 (Abcam, USA; #ab175408), anti-CYP17A1 (Abcam, USA; #ab125022) and anti-STAR (Abcam, USA;#ab203193). After then the membranes were rinsed with TBST three times (10 min each) and then incubated with secondary antibody (1:5000) labelled with horseradish peroxidase (HRP) at room temperature for 1 h. After the reaction the membranes were rinsed with TBST three times (10 min each), the signals were detected using the enhanced chemiluminescence Western blot kit (EzWestLumi plus) from ATTO Corporation (Motoasakusa, Tokyo, Japan) and analyzed using the ImageQuant LAS 4000 mini (GE Healthcare) machine. The experiments were performed in triplicate and data were quantified by Image J program.