Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g

Western blotting was performed according methods previously described.17 Briefly, total protein was isolated using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors (Beyotime Biotechnology, Shanghai, China). The primary antibodies used were: anti‐HIF‐1α (#36169, 1:1000), anti‐caspase‐3 (#9662, 1:1000), and anti‐GAPDH (#5174, 1:1000, Cell Signaling Technology, Danvers, MA, USA); and anti‐NDUFA4L2 (A14288, 1:1000, ABclonal, Woburn, MA, USA). Horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulin G (1:1000, Beyotime Biotechnology) was used as a secondary antibody. All experiments were repeated at least three times.

Total protein samples were available from lysed lung tissues using sodium dodecyl sulfate (SDS; Beyotime, Shanghai, China) containing proteinase inhibitors and determined by bicinchoninic acid assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). SDS-polyacrylamide gel electrophoresis (10%) was applied to separate various protein bands, and all the bands were visible on polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Then the membranes were incubated with primary antibodies anti-B-cell lymphoma-associated X protein (Bax), B-cell lymphoma (Bcl)-2, caspase-3, caspase-9 (1: 1000; Abcam, Cambridge, UK), and anti-tubulin (1: 1000, CST, Fall River, MA, USA) at 4°C overnight. Then the clean membranes were cocultured with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Beyotime) for 2 h at room temperature. Finally, a specific band was enhanced with chemiluminescence reagent (ECL, Thermo) and was visible through the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA)

m⁶A dot blots were conducted as previously described with some modifications (54 (link)). Ploy(A)⁺ mRNA samples were denatured at 50°C for 15 min in three sample volumes of RNA incubation buffer. An equal volume of chilled 20x SSC buffer (Sigma-Aldrich) was then added before samples were spotted on a polyvinylidene difluoride (PVDF) membrane (Millipore) and fixed at 80°C for 40 min. The membrane was blocked with 5% nonfat milk and incubated with anti-m⁶A antibody (1:10,000; Synaptic Systems) overnight at 4°C. Then, horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (Beyotime) was added to the blots for 1 hour at room temperature, and the membrane was developed with Amersham ECL Prime Western Blotting Detection Reagent (Millipore).