Hrp conjugated anti rabbit igg sa00001 2

Western blotting was performed as described previously.³¹ Protein was obtained at the indicated times using lysis buffer containing proteinase inhibitors (87785, Thermo Fisher Scientific). The obtained supernatant was separated via 10% SDS‐PAGE gels (Takara, Japan) and transferred to PVDF membranes (Millipore, USA). Following blocking by 10% milk for 40 min at RT, the proteins were incubated overnight at 4°C with anti‐NSUN2(20854, Proteintech), anti‐PFAS (76957, CST), anti‐GAPDH (60004‐1‐Ig, Proteintech), anti‐β‐actin (66009‐1‐Ig, Proteintech) and anti‐ALYREF (ab202894, Abcam). After incubated with HRP‐conjugated anti‐rabbit IgG (SA00001‐2, Proteintech) or HRP‐conjugated anti‐mouse IgG (SA00001‐1, Proteintech) at RT for 1.5 h. Subsequently, signal detection was performed using an ECL kit and visualized with the detection instrument.

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).