Coomassie plus bradford reagent

Transduced cells were lysed using M-PER lysis buffer with 5 mM EDTA and HALT protease inhibitor cocktail (all Thermo Scientific) and cleared by centrifugation at 14,000g at 4°C for 10 min. Protein concentrations were determined using Coomassie Plus Bradford reagent (Thermo Scientific) and equal amounts of protein were prepared using Laemmli buffer (Bio-Rad) with added DTT. After denaturation at 95°C for 5 min, proteins were separated on 4%–20% Mini-Protean TGX gels (Bio-Rad) and blotted onto PVDF membranes using the TransBlot Turbo Transfer system (Bio-Rad). Membranes were blocked with blocking buffer (5% milk, 5% FBS, 1% BSA, 1 M Glycine) at room temperature and probed with primary antibodies at 4°C overnight. Primary antibodies were α-ADAR1 (sc-73408, Santa Cruz Biotechnology; 1:500), α-ADAR2 (sc-73409, Santa Cruz Biotechnology; 1:1000), and α-GAPDH (MAB374, Millipore; 1:25,000). Membranes were probed with α-mouse HRP secondary antibody (P0447, DAKO, Glostrup, Denmark) for 1 h at room temperature, and chemiluminescence was detected using Immobilon Forte Western HRP substrate (Millipore) on an Amersham Imager 600 (GE Healthcare).