Ab126745

A total of 5–10 × 10⁶ cells were harvested in RIPA buffer for total protein extraction. Overall, 10–20 μg of each sample were loaded onto 10% polyacrylamide gels for gel electrophoresis. Primary antibodies (polyclonal anti-ADAR1 ab88574 1:500, monoclonal anti-ADAR1 ab126745, 1:1000, and anti-actin ab8227, 1:1000, Abcam) were prepared in blocking buffer. Membranes were incubated overnight at 4 °C with primary antibodies, followed by secondary antibody incubation (anti-rabbit HRP or anti-mouse HRP, 1:5000, Cell Signaling Technology; or anti-chicken HRP, 1:5000, Abcam) in blocking buffer for 1 h at room temperature. Blots were developed using enhanced chemiluminescence (Femto Detection kit, Promega) on a Chemidoc digital imaging machine. Representative images out of three independent Western blots are shown in main and supplementary figures, with uncropped images of key blots provided in Supplementary Fig. 5. Nanofluidic experiments were performed with the Nanopro 1000 instrument (Cell Biosciences). ADAR1 was detected using an ADAR1-specific antibody (ab168809, 1:500 Abcam). A β2-microglubulin-specific antibody (β2M; 1:500 Upstate) was used to normalize the amount of loaded protein.

Cells or frozen tissues were lysed in RIPA buffer and proteins (60 μg) were separated on 10–12% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking, membranes were incubated with appropriate dilutions of specific primary antibodies followed by incubation with HRP-conjugated secondary antibodies and visualization using ECL system (Thermo Fisher Scientific, Rochester, NY, USA). Anti-β-ACTIN (1 : 1000, c4) and anti-AR (1 : 1000, N20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ADAR1 (1 : 1000, monoclonal, ab126745) antibody, anti-ADAR1 (1 : 1000, polyclonal, ab88574), anti-QKI (1 : 1000, ab126742) were purchased from Abcam (Shanghai, China). Anti-ADAR2 (1 : 500, ENT0119) antibody was purchased from Elabscience Biotechnology (Bethesda, MD, USA).

Human gastric cancer and adjacent gastric mucosa specimens were collected from 38 patients at Shanghai East hospital. Written informed consent was obtained from all participants and study protocol was approved by the ethics committee of Shanghai East Hospital, Tongji Univeristy School of Medicine. After resection, all the samples were snap-frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Human gastric cancer tissue microarray (ST2091) containing 208 patients’ sample was purchased from Xian Alenabio Company. Among these patients, 64 were women and 144 were men, with an age range between 17 and 84 years old. ADAR1 expression was assessed by immunohistochemical staining using an anti-ADAR1 antibody (ab126745, Abcam) at a dilution of 1:500. The staining results were classified according to the percentage of positive cells and staining intensity: negative (−), slight positive (+), moderate positive (++), or strong positive (+++). To prevent bias from knowledge of clinical data, scoring of staining intensity was conducted in a blinded manner.