All processes were based on an existing protocol [28 ]. For this assay, total RNA from PTECs (5 × 106) was isolated using TriZol (15596-026, Invitrogen, USA), incubated at room temperature for 5 min. Thereafter, polyadenylated mRNA was purified from the extracted total RNA. The concentration of RNA was determined using a spectrophotometer (ND-2000, ThermoFisher, USA), and the quantity of RNA sample was checked via electrophoresis on a 0.8% agarose gel (16500-500, Invitrogen, USA). Next, RNA samples were prepared and loaded with the wells. The wells were then covered with foil to prevent light, and electrophoresis was run at 35 V overnight.
For the m6A content quantification test, after confirming that the RNA samples were transferred from the gel to the membrane, the membrane was blocked in 5% nonfat milk (P0216, Beyotime, China) in TBST at room temperature for 1 h, rinsed in TBST for 15 min, and incubated with the primary antibody (Abcam, UK) against m6A (ab284130) or IgG (ab172730) at 4 °C overnight and with the secondary antibody against rabbit IgG at room temperature for 1 h. Further, BeyoECL Plus reagent was applied to the membrane, and the m6A content was calculated using ImageJ 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
For the m6A content quantification test, after confirming that the RNA samples were transferred from the gel to the membrane, the membrane was blocked in 5% nonfat milk (P0216, Beyotime, China) in TBST at room temperature for 1 h, rinsed in TBST for 15 min, and incubated with the primary antibody (Abcam, UK) against m6A (ab284130) or IgG (ab172730) at 4 °C overnight and with the secondary antibody against rabbit IgG at room temperature for 1 h. Further, BeyoECL Plus reagent was applied to the membrane, and the m6A content was calculated using ImageJ 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).