Anti fto antibody

After lysed with RIPA buffer and the protein sample (20 μg/band) were separated with 1×SDS-PAGE, protein bands transferred into the PVDF membrane (Merck Millipore) were detected with the primary antibodies (anti-METTL3 antibody (1:1000), anti-FTO antibody (1:1500, Abcam) first, then HRP-bound secondary antibody. The membrane was visualized with Chemiluminescence Detection Kit (Servicebio, China) and detected under a Bio-Rad imaging system (Bio-Rad, USA).

Total proteins were isolated from tissues and cells using RIPA lysis buffer mixed with phenylmethylsulfuryl fluoride (PMSF) (Bioss, China) and quantified using the bicinchoninic acid (BCA) method (Beyotime, China). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking by 5% skim milk, the PVDF membranes were incubated with an anti-FTO antibody (1:1 000, Abcam, USA), anti-CDK6 antibody (1:1 000, Abcam, USA) and anti-GAPDH antibody (1:5 000, Bioss, China) at 4 °C overnight. The PVDF membranes were then incubated with a secondary antibody (1:10 000, Boster, China) for 1 h at room temperature. The proteins were measured using the AMERSHAM ImageQuant 800 system (USA).

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.

After the neurons were washed with PBS, RIPA lysis buffer (Merck Millipore, Cat. No. 20–188) containing protease inhibitors (Roche, Cat. No. 5,892791,001) was added to fully lyse the cells on ice for 30 minutes, followed by centrifugation at 12,000 X g at 4°C for 10 minutes. The supernatant was collected, and the PierceTM BCA Protein Assay Kit (Thermo Scientific, Cat. No. 23225) was used to determine the protein concentration. Finally, the protein was denatured at 100°C for 10 minutes. 20ug of protein were analysed by immunoblotting using an anti-Mettl3 antibody (Abcam Cat. No. ab195352), an anti-Fto antibody (Abcam Cat. No. ab280081), with an anti-β-actin antibody (Abcam Cat. No. ab8226) used as an internal control.

Western blotting was performed as described previously (Shi et al., 2015 (link)). Specifically, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Antibodies for detecting protein were diluted at 1:1,000 for anti-METTL3 antibody (Abcam), anti-FTO antibody (Abcam), an anti-NRIP1 antibody (Abcam), an anti-METTL3 antibody (Abcam), an anti-NRIP1 antibody (Abcam), and 1:5,000 for anti-β-ACTIN antibody (Abcam). The horseradish peroxidase–labeled secondary antibody (Abcam) was diluted at 1:10,000 and detected with enhanced chemiluminescence NcmECL Ultra kit (NCM Biotech, China).