Adar2

Total protein extracts were isolated with RIPA lysis buffer in the presence of a protease inhibitor mixture and phosphatase inhibitor cocktail (SIGMA). Protein extracts were quantified with the BCA Protein Assay Kit (Pierce). Equal amounts of total cellular lysates (30 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, analyzed by immunoblotting with the appropriate antibodies and then revealed by ECL (GE Healthcare). The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).

Total protein extracts were obtained by lysing the cells with hot Laemmli buffer (60 mM Tris-HCl pH 6.8, 100 mM DTT, 5% glycerol, 1.7% SDS) and passed through syringes (26G) [3 (link)]. Cytosolic and nuclear protein extracts were isolated from mesothelioma cells, and their purity was assessed as previously described [20 (link)]. A total of 5 μg of protein extract was separated on denaturing 15% SDS-PAGE gels, and proteins were transferred onto PVDF membranes (0.45 μm, Perkin Elmer, Waltham, MA, USA). Membranes were probed with the following primary antibodies: RBM8A (Sigma-Aldrich, St. Louis, USA, cat. #HPA018403, RRID:AB_1858908), ADAR1 (Sigma-Aldrich, cat. no. HPA003890, RRID:AB_1078103), ADAR2 (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc-73409, RRID:AB_2289194), MSI (Cell Signaling Technology, Danvers, USA, cat. no. 85652, RRID:AB_2800060), and mouse anti-β-actin (C4, MP Biomedicals MP691002 RRID:AB_2335127). Membranes were then incubated with one of the following secondary antibodies: rabbit anti-mouse IgG-HRP (no. A9004) or goat anti-rabbit IgG-HRP (no. A0545), obtained from Sigma Aldrich. The signals were detected by enhanced chemiluminescence (Clarity TM ECL Substrate, BioRad, Hercules, CA, USA) using Fusion Digital Imager (Vilber Lourmat, Marne-la-Vallée, France). Quantification was carried out using ImageJ software, version 1.52a.