Anti cyp11a1

Mice received i.p. injections of insulin (1 U/kg) or 0.9% saline and were euthanized 20 min later by cervical dislocation. Tissues were extracted and immediately snap-frozen in liquid nitrogen. Homogenized tissue samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After blocking with 5% milk dissolved in Tris-buffered saline containing 0.05% Triton X-100 (TBS-T), the membranes were incubated with anti-phospho-Ser473 Akt, anti-Akt, anti-phospho-Thr183/Tyr185 JNK/SAPK, anti-JNK/SAPK, anti-StAR antibody, anti-Cyp11A1, anti-FAS, anti-α-tubulin, or anti-β-actin antibodies (Cell Signaling Technology; Beverly, MA, USA) (1:1000) overnight at 4 °C. The next day, the membranes were washed with TBS-T and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000). After washing with TBS-T, the blots were developed using an Immobilon chemiluminescent HRP substrate (Merck Millipore; Darmstadt, Germany). The blot images were scanned and analyzed using Image J software (National Institutes of Health; Bethesda, MD, USA).