Hybond n membrane

Total RNAs were harvested from pericytes or retinal vessels using the Trizol reagent. mRNAs were enriched using the Dynabeads mRNA Direct Purification Kit. RNA quantity was monitored by the NanoDrop ND-1000 Spectrophotometer (Agilent, USA). mRNAs were denatured by heating at 95°C for 5 min and then chilled on the ice. Then, mRNAs were blotted onto Hybond N⁺ membranes (FFN13, Beyotime, China). UV crosslinking of RNAs to the membranes were conducted in an Ultraviolet Crosslinker. The membranes were blocked with 5% defatted milk for 1 h at room temperature. The membranes were incubated with m⁶A-specific antibody (1:1000, 202003, Synaptic Systems) at 4°C overnight. After three washes with 1 × PBST, the membranes were incubated in HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2500, sc-2030, Santa Cruz) for 1 h at room temperature with gentle shaking. Finally, the membranes were washed again three times in 1 × PBST. The signals were detected by the SuperSignal West Dura Extended Duration Substrate (34075, Thermo Fisher Scientific). Quantified m⁶A levels were normalized to the amount of the loaded mRNAs.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the standard manufacturer's protocol and then quantified and diluted in 10 mM Tris‐EDTA buffer. The indicated amounts of RNA samples were loaded onto Hybond‐N + membranes (FFN10, Beyotime, China). The membrane was crosslinked at 254 nm UV for 60 s after a short drying process, blocked with 5% milk for 1.5 h at RT and incubated with an anti‐m⁵C antibody (ab214727, Abcam, USA) at 4°C overnight. After three washes with TBST (Thermo Fisher Scientific, USA), the membranes were incubated with HRP‐conjugated anti‐rabbit IgG (SA00001‐2, Proteintech) for 1.5 h at RT and then visualized using an enhanced chemiluminescence kit (WBKLS0100, Thermo Fisher Scientific) and a detection instrument (Tanon Science, Shanghai, China).