Amersham hybond p pvdf transfer membrane

For Western blotting, total cell lysates were obtained using NP40 buffer (0.1% Nonidet P-40 (NP-40), 0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). For the isolation of cytosolic fractions, cells were lyzed in buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.15% NP-40, 1 mM DTT) with protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 12,000 g for 30 seconds at 4°C, and supernatants were taken as cytosolic fraction. The pellets were washed with ice-cold phosphate-buffered saline (PBS) and lyzed in buffer B (20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 1 mM DTT). After sonication (5 seconds at 50 watt), samples were centrifuged at 12,000 g for 15 minutes at 4°C, and supernatants were taken as nuclear fractions. Protein concentration was determined using Bradford assay (Biorad). Thirty micrograms of protein was separated using SDS-PAGE and transferred to Amersham Hybond-P PVDF Transfer Membrane (GE Healthcare; RPN303F). Membranes were blocked in 5% milk in Tris-buffered saline-0.01% Tween20 and incubated with the indicated antibodies. Membranes were visualized using a ChemiDoc XRS+ System (Bio-Rad) using Image Lab software version 5.2.1 (Bio-rad). Images have been cropped for presentation, but full-size images are presented in S4 Fig.