Nih image

Sertoli cells cultured in six multiwell plates were washed once with PBS at room temperature. Then, 200 µL PBS containing 2 µL protease inhibitor cocktail Sigma-Aldrich (P-8340), 1 mM NaF, 1 mM EGTA, 1 mM EDTA, 50 nM okadaic acid and 2 mM PMSF were added to each well and cells were disrupted by ultrasonic irradiation. A 200 µL volume of 2× Laemmli buffer was added and thoroughly mixed. Proteins were resolved in 5% or 10% SDS-PAGE in a Mini Protean 3 Cell (Bio-Rad). After SDS-PAGE, gels were equilibrated in transfer buffer for 10 min and electrotransferred at 100 V for 60 min onto PVDF membranes (Hybond-P; Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK) using a Mini Trans-Blot Cell (Bio-Rad). Membranes were probed with commercial antibodies that recognize the phosphorylated forms of p70S6K (Thr389) and ACC (Ser79), with antibodies against the non-phosphorylated form of these proteins (Cell Signaling Technology) and with antibodies against Cyclin D1 (Abcam), Cyclin D3 and AKT (Cell Signaling Technology). 1:1000 dilutions of primary antibodies were used. The intensities of the autoradiographic bands were estimated by densitometric scanning using NIH Image (Scion Corporation, Frederick, MD, USA) software.