2.5. Levels of Steroidogenic Enzymes

The levels of nine key steroidogenic proteins were measured by western blot. Cells were cultured in 6-well plates at 500,000 cells/well. After attaching to the plate for 24 h, the cells were exposed to 10 and 100 µM DBP, 100 and 500 µM MBP, or solvent control (0.1% DMSO) dissolved in a culture medium with 0.1 mM dbcAMP. After 48 h exposure, the cells were washed with DPBS and trypsinized for 10 min. The detached cells were then collected in tubes and lysed with RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, 1% protease inhibitors pH 8). Protein concentration in the lysate was determined by the Lowry assay [22], and 20 µg of protein per sample was separated by SDS-PAGE on a 4–20% gel before being transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Total protein content in each lane was measured by staining with a No-Stain™ Protein Labeling Reagent kit according to the manufacturer’s protocol. The membranes were then blocked for 1 h in TBS with 5% dry milk and incubated overnight with primary antibodies (Table 1), diluted in Superblock™ solution. After the incubation, the membranes were washed twice with TBS-Tween (TTBS) and twice with TBS. Secondary antibody (peroxidase-conjugated goat anti-rabbit or goat anti-mouse (Cat. no. 5196-2504 and 0300-0108P respectively, Bio-Rad, Hercules, CA, USA)) diluted 1:5000 in Superblock™ was then added, and the blots were incubated for 1 h, followed by washes as described above. The blots were developed with an ECL-kit using a charge-coupled device (CCD) imager (iBright 1500 Imaging System; ThermoFisher, Rockford, IL, USA), and the optical density for the blots and total protein staining were measured with ImageJ ver 1.53r (NIH, Bethesda, MD, USA). The results were normalized against the total protein content for each sample.

List of primary antibodies used for western blot analysis.