Ecltm western blotting detection reagent

For Western blot analysis, samples were immediately frozen with liquid nitrogen and stored at −25°C. The SG (SLG + SMG and PG), Adr, and placenta homogenates (each n = 3) were suspended in 0.25 M sucrose solution (200 μL) and then centrifuged at 900 × g for 10 min at 4°C. After protein concentrations were estimated using the Lowry protein assay (35 (link)), the protein concentration of each sample was adjusted to 1 μg/μL and 20 μg total protein samples were applied. The homogenates were separated by SDS-PAGE and then transferred onto PVDF membranes (ATTO, Tokyo, Japan) for Western blot analysis. After blocking with 3% skim milk at room temperature for 1 h, the PVDF membranes were incubated for 2 h with the primary antibodies (anti-GAPDH, 1:400; anti-CYP11A1, 1:400; anti-StAR, 1:200; anti-steroid sulfatase, 1:200 and anti-CYP11β1, 1:500) at room temperature. After four washes with PBS for 15 min each, the membranes were incubated with secondary goat anti-rabbit IgG (1:2,000) at room temperature for 2 h. After four washes with PBS for 15 min each, the membranes were reacted for 1 min with ECL^TM Western blotting detection reagents (Amersham Biosciences, England). The expression level of each protein was determined densitometrically using CS Analyzer, ver. 3.0 (ATTO).

All fractions were analysed by SDS-PAGE and immunoblotting as described in Kebbi-Beghdadi et al. [36 (link)]. The mouse polyclonal anti_Wcw_1706 antibody diluted 1/1000 was incubated overnight at 4 °C in TBS with 0.05% Tween 20 and 5% non-fat dry milk. Mouse monoclonal anti-GST (Sigma-Aldrich, Buchs, Switzerland) and anti-His (Sigma-Aldrich, Buchs, Switzerland) antibodies were diluted respectively 1/1000 or 1/3000 and incubated during 2 h at room temperature in TBS with 0.05% Tween 20 and 0.5% non-fat dry milk. Secondary goat anti-mouse IgG-HRP (BioRad, Cressier, Switzerland) antibody (1/3000 dilution) was applied during 1 h at room temperature. Immunoblots were revealed with ECL^TM Western Blotting Detection Reagent (Amersham, GE Healthcare, Glattbrugg, Switzerland) and analysed on ImageQuant LAS4000 mini (Amersham, GE Healthcare, Glattbrugg, Switzerland).

Cells were harvested and lysed using lysis buffer (Cell Signaling Technology) containing protease inhibitors (Sigma) and phenylmethylsulfonyl fluoride (PMSF) (Sigma Chemical Co., St Louis, MO, USA). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to Immobilon-P Transfer Membranes (Millipore Corporation, Bedford, MA, USA). The membranes were pre-blotted in 5% skim milk prior to incubation in 1% skim milk containing primary anti-Sp1 (1:500; Millipore Darmstadt, Germany), anti-KLF12 and anti-XIAP (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-cleaved-PARP, anti-cleaved-caspase3 (1:1000; Cell Signaling Technology, Inc., Danvers), anti-DDK (1:1000) (OriGene Technologies, Rockville, MD) and anti-β-actin (1:10000; Sigma-Aldrich, St. Louis, MO) antibodies overnight. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Amersham) and visualized using ECL^TM Western Blotting Detection Reagent (Amersham). Images were captured by Fuji Medical X-Ray Film (Fuji) and developed by the Fuji system. The Human Apoptosis Array Kit (R&D Systems, Inc., USA) was used based on the manufacturer’s instructions.