Rabbit anti m6a

The primary and secondary antibodies were purchased from commercial sources as follows: Mouse anti-FTO, Mouse anti-Mad2, Mouse anti-Cdc20, Mouse anti-Bub1, Mouse anti-Bub1b, Mouse anti-Bub3, Mouse anti Tubulin (Santa Cruz Biotechnology), Rabbit anti m⁶A (Synaptic Systems), Rabbit anti-Actin (Sigma-Aldrich). HRP-goat anti rabbit IgG (CWbio) and HRP-goat anti mouse IgG (CWbio).

RNA m⁶A dot blotting was employed to verify m⁶A binding with anti-m⁶A antibody. HT22 cells were collected at 95% confluence, and total RNA was isolated and purified according to the standard protocol of the Total RNApure Kit (#ZP404-1, ZOMANBIO, China). The 6 μg RNA suspended in 200 μL RIP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with 0.2 μL RNase inhibitor (#N8080119, Thermo Fisher Scientific), and incubated overnight at 4 °C with magnetic beads bound with 5 μg of rabbit anti-m⁶A (#202003, Synaptic Systems, Germany) or rabbit anti-IgG (#AC005, ABclonal, China) antibodies. After IP, the aliquots of sample solution were then detected by dot blotting analysis to verify m⁶A binding. Briefly, the aliquots above were added to the nitrocellulose membrane (#66485; Biosharp, China). The membrane was then cross-linked by UV light, sealed for 30 min, and incubated overnight at 4 °C in rabbit anti-m⁶A antibody (1:1000, #202003, Synaptic Systems, Germany). After that, the membrane was washed in TBST thrice for 5 min and further incubated for 2 h in corresponding Dylight™ 800 conjugated anti-rabbit IgG (H&L) (GOAT) antibody (1:10,000, #611–145-002, ROCKLAND, USA) at room temperature (~25 °C). After washing thrice again in TBST, the reactive bands were visualised using an Odyssey IR fluorescence scanning imaging system (LICOR, USA).