Sc81852

Cells were trypsinized, counted, washed with ice-cold PBS and lysed in Laemmli buffer (50 mM Tris-HCL, 2.5 mM EDTA, 2.55 mM EGTA, 2% SDS 20%, 5% Glycerol, 1% Bromophenol blue, protease inhibitor cocktail tablets and 2 mM DL-Dithiothreitol solution) at 10 million cells/ml. Protein lysates were sonicated and denatured at 95 C for 5 min and electrophorated on 4-15% Mini-PRO-TEANÒTGXTM gels (456-1084, BIO-RAD), transferred onto nitrocellulose membranes (1704159, BIO-RAD). Membranes were incubated overnight at 4 C with mouse anti-STAG2 (1:1,000, Santa Cruz Biotechnology, sc-81852), goat anti-STAG1 (1:5,000, ab4457, Abcam), rabbit anti-H3 (1:50,000, ab1791, Abcam), mouse anti-b-Actin (1:20,000, A5316, Sigma-Aldrich), rabbit anti-FLI1 antibody (1:1,000, ab133485, Abcam) and rabbit anti-H3K27ac (1:1,000, ab4729, Abcam). Then membranes were incubated 1h at room temperature with respective anti-rabbit, anti-mouse immunoglobulin G horseradish peroxidase (HRP) coupled secondary antibody (1:3,000, NA934 or NXA931, respectively; GE Healthcare) or anti-goat IgG-HRP (1: 10,000, SC-2354, Santa Cruz). Proteins were visualized using SuperSignalÔ West Pico Plus (34580, Thermo Scientific) and ChemiDocÔ Imaging System (BIO-RAD).