Horse radish peroxidase conjugated anti rabbit immunoglobulin g igg

The ${\mathrm{m}}^6\mathrm{A}$ dot blot assay is a classic and relatively straightforward method to detect global ${\mathrm{m}}^6\mathrm{A}$ differences in total RNA.^{41 (link)} Total RNA from RAW264.7 cells and human PBMCs was extracted via TRIzol (Cat. #15596018, Invitrogen). Then the total RNA was diluted to 1,000, 800, 500, 400, 250, and $200\text{\hspace{0.17em}ng}/\mathrm{\mu }\mathrm{L}$ , serially diluted, and spotted on a nylon membrane (Cat. #FFN13, Beyotime Biotechnology). The membrane was cross-linked at ultraviolet (UV) $254\mathrm{\hspace{0.17em}nm}$ , $0.12{\mathrm{J}/\mathrm{cm}}^2$ . After UV cross-linking, the blotted membrane was blocked in PBS containing 0.1% Tween-20 (PBST; Cat. #30189328, Sinopharm Chemical Reagent Company) and 5% nonfat milk (Cat. #FD0080, FDbio Science) for 1 h at room temperature. The membrane was then incubated with ${\text{anti-m}}^6\mathrm{A}$ antibody (1:1,000 dilution; Cat. #ab284130, Abcam) at 4°C overnight. The membrane was incubated with horseradish peroxidase (HRP)–conjugated antirabbit immunoglobulin G (IgG; 1:5,000 dilution; Cat. #7074, Cell Signaling Technology) at room temperature for 1 h and visualized by FDbio-Dura ECL Kit (Cat. #FD8020, FDbio Science). Dot blot analysis of RNA from RAW264.7 cells was performed three independent times ( $n=3$ biological replicates).