Donkey anti mouse or donkey anti goat secondary antibodies

Cells were washed twice in PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % NP-40, 1 mM Sodium Orthovanadate, 1 mM NaF) containing Halt protease inhibitors Cocktail (Thermo Scientific, ref 87786). Lysates were sonicated using the Bioruptor® (Dia-genode) and cleared by centrifugation at 16,000g for 20 min at 4 °C. Total protein lysates were separated by SDS-PAGE in 8% polyacrylamide gels and transferred to nitrocellulose membranes. Subsequently, membranes were washed with TBS-T (50 mM TRIS + 150 mM Sodium chloride + 0,1% Tween 20, pH 7,4) and blocked using 5% non-fat milk solution as blocking agent in TBS (50 mM TRIS + 150 mM Sodium chloride) for 1 h at RT. Membranes were then incubated with primary antibodies anti-ARID2 (E-3, sc-166117, Santa Cruz) and anti-Actin (I-19, sc-1616, Santa Cruz), diluted 1:200 and 1: 1,000 in TBS-T/5% (w/v) BSA at 4°C overnight, respectively. Donkey anti-mouse or donkey anti-goat secondary antibodies (LI-COR Biotechnology, Lincoln, USA) conjugated to IRDye 800CW (926-32212) or IRDye 680RD (926-68074) respectively were used as secondary antibodies and visualized using Odyssey Clx imager (LI-COR Biotechnology, Lincoln, USA).