2.7 Total protein extract and western blotting assay
Briefly, placental, adrenal tissue,
and NCI-H295R cells were rinsed three times with ice-cold
phosphate-buffered saline (PBS) and then lysed for 30min at 4°C in
radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase
inhibitor cocktail, followed by the BCA Assay kit for protein
quantification. A total of 30μg of proteins was loaded to each lane,
isolated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). For the transfer to the membrane, a wet
transfer was performed using an assembly of polyvinyl diisopropyl
fluoride (PVDF) membranes (Millipore, MA, USA). The transfer was
performed at 100v, 350 mAmp for 1h. After three washes with TBST (25 mM
Tris-HCl, 50 mM NaCl, 0.05% Tween-20), the membrane was blocked with
5% skim milk in TBST for 1h. The primary antibodies, including anti-P
Glycoprotein (P-gp) (1:1000 dilution, Abcam, ab170904) and
anti-hydroxysteroid 11beta- dehydrogenase 1, (11β-HSD1) (1:1000
dilution, Abcam, ab39364), anti-11β-HSD2 (1:1000 dilution, Santa Cruz,
sc-365529), anti-Sirt3 (1:1000 dilution, ABclonal, A7307), anti-IGF1
(1:200 dilution, Santa Cruz, sc9013) and anti-steroidogenic acute
regulatory protein (StAR) (1:1000 dilution, ABclonal, A16432) were added
to TBST in 5% skim milk and placed on constant shaking at 4°C
overnight. After incubation and washing, the membranes were incubated
with secondary antibodies (anti-mouse and anti-rabbit conjugated with
horseradish peroxidase) at a 1:5000 dilution for 1 h and were detected
using an enhanced chemiluminescent detection kit (Bridgen, Beijing,
China; D046). Densitometric analyses were performed using ImageJ
software (National Institutes of Health).