Chemiluminescence detection kit

The cells were directly lysed in 1× SDS-PAGE loading buffer. Protein bands were detected sequentially with primary and HRP-bound secondary antibodies, visualized using a Chemiluminescence Detection Kit (Servicebio, Wuhan, China), and detected with an imaging system (Bio-Rad, USA). Antibodies against METTL3 (AB195352, 1:2000) were obtained from Abcam. GAPDH (60004, 1:5000) antibodies were purchased from Proteintech.

The cells were directly lysed in 1× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and subjected to SDS-PAGE. The blots were sequentially incubated with primary and HRP-conjugated secondary antibodies. The immunoreactive bands were then visualized using a Chemiluminescence Detection Kit (Servicebio, Wuhan, China) and detected with an imaging system (Bio-Rad, USA). Antibodies against FTO (1:2000, RRID:AB_280081) and caveolin-1 (1:1500, RRID: AB_32577) were obtained from Abcam. Antibodies against Pink1 (1:1500, Cat#6946), MFN2 (1:1000, Cat#11925), p-Parkin1 (1:1500, Cat#36728), Parkin1 (1:1000, Cat#2131), Cytochrome c (1:2000, Cat#12963) and β-tubulin (1:1500, Cat#2146) were obtained from Cell Signaling Technology. Antibody against p-MFN2 (1:3000, Cat#ABC963) was obtained from Merck Millipore. Antibody against GAPDH (1:5000, Cat# 60004) antibodies was purchased from Proteintech.

Total protein from the GCs was extracted using RIPA reagent (Servicebio, China), denatured, separated on a 12% polyacrylamide SDS-PAGE gel, and transferred to a nitrocellulose membrane. After blocking, the blots were incubated with antibodies against YAP1 (1:1000, 14074S, CST, USA) and β-actin (1:1000, 66009-1-Ig, Proteintech, China) in Tris-buffered saline (TBS) containing 0.1% Tween-20 at 4 °C, overnight. The following day, the blots were incubated with a goat anti-rabbit or anti-mouse horseradish peroxidase immunoglobulin G (H+L) secondary antibody (1:5000, SA00001-2, Proteintech, China) at 37 °C for 2 h. The protein bands were detected using a chemiluminescence detection kit (Servicebio, China) on X-ray films and analyzed using Quantity One software (Bio-Rad, USA).