Anti alkbh5 antibody

Total protein was extracted from the frozen clinical tissues, murine model tissues or cells with radioimmunoprecipitation assay (RIPA) Lysis Buffer and PMSF (Beyotime, Shanghai, China). The protein was electrophoresed on 7.5%-12% SDS–polyacrylamide gels according to the different proteins molecular weight and transferred to nitrocellulose membranes (Bio-Rad, Hercules, USA). The membranes were incubated with the anti-IL-17RA receptor antibody, anti-FTO antibody, anti-ALKBH5 antibody (Abcam, Cambridge, USA), anti-METTL3 antibody (Zen bio, China), anti-METTL14 antibody (Sigma-Aldrich, Germany), β-Actin antibody (Proteintech, USA) overnight at 4°C. And then the membranes were incubated with secondary antibody IRdye 680RD goat (polyclonal) anti-mouse IgG (H+L) and IRdye 800CW goat (polyclonal) anti-rabbit IgG (H+L) (Licor Biosciences, Nebraska, USA) for 2 hours at room temperature. The membranes were detected using an Odyssey infrared scanner (Licor Biosciences, Nebraska, USA). The antibodies are listed in Table S3.

Cell samples were fixed with paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) (4% in phosphate buffered saline solution [PBS]), blocked and permeabilized with 3% bovine serum albumin (BSA; Sigma-Aldrich) and 0.5% Triton X-100 (Sigma-Aldrich) in PBS, and incubated overnight at 4°C with the rabbit anti-ALKBH5 antibody (1:100; Abcam Ltd., Cambridge, UK) which were diluted with 1% BSA. After rewarming for one hour at room temperature, samples were incubated with the Alexa Fluor 488 labeled second antibody (1:200; Invitrogen-Gibco) for four hours at 37°C. Then, the nuclei were labeled with Hoechst (1:2000, Sigma-Aldrich). Samples were washed three times with PBS. The images were finally captured using a confocal microscope (SP8, Leica, Wetzlar, Germany).

Protein was extracted from the rat lung tissues with lysis buffer (RIPA: PMSF=100:1), and equal amounts of protein from each sample (20 μg or 40 μg) were separated by 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with high-affinity anti-YTHDF1 antibody (1:1000, Proteintech), anti-YTHDF2 antibody (1:5000, Proteintech), anti-METTL3 antibody (1:1000, Abcam), anti-METTL14 antibody (1:1000, Bioss Antibodies), anti-FTO antibody (1:1000, Abcam), anti-ALKBH5 antibody (1:1000, Abcam), anti-WTAP antibody (1:5000, Proteintech), anti-VIRMA antibody (1:1000, Abcam), anti-RBM15 antibody (1:1000, Proteintech), anti-YTHDF3 antibody (1:1000, Abclonal), anti-YTHDC1 antibody (1:1000, Proteintech), anti-YTHDC2 antibody (1:1000, Abclonal), anti-IGF2BP1 antibody (1:1000, Abcam), anti-IGF2BP2 antibody (1:1000, Abcam), anti-IGF2BP3 antibody (1:1000, Abcam), and anti-β-actin antibody (1:5000, Proteintech), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:5000, Proteintech). The chemiluminescent signals were detected with a chemiluminescent HRP substrate [51 (link)]. Densitometric analysis was conducted with ImageJ software.