Rabbit anti mettl3

A cell climbing slice was prepared in a 24-well plate. After attaching to the wall, the cells were rinsed for 5 min with PBS buffer three times and then fixed in 4% formaldehyde at 4°C for 10 min. The serum was diluted to 5% with room-temperature PBS and incubated at room temperature for 30 min (closed liquid containing 0.1% Triton X-100). The primary antibodies used included rabbit anti-METTL3 (dilution, 1 : 200; Abcam) and mouse anti-PININ (dilution, 1 : 100; PININ). After being rinsed gently with PBS three times, the cells were incubated at 37°C for 1 h with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (dilution, 1 : 1000; Invitrogen) and Alexa Fluor® 568-conjugated goat anti-mouse IgG (dilution, 1 : 1000; Invitrogen) as secondary antibodies. The cells were viewed under an N-STORM superresolution system (Nikon).

The tissues and cells of each group were lysed in RIPA solution (Beyotime, Shanghai, China) for 30 min and centrifuged at 14000 RPM and 4 °C, and the supernatant collected. The protein concentration was determined by the BCA method (Pierce, Rockford, IL, USA). The proteins were separated in 4% or 10% concentrated gel and transferred to a PVDF membrane. The PVDF membrane was blocked by placement in 5% skimmed milk powder at room temperature for 1 h. Next, the membranes were incubated at 4 °C with rabbit anti-METTL3 (1:1000, Abcam) or rabbit anti-CCNG2 (2.5 µg/mL, Sigma, St. Louis, MO, USA) overnight. After washing with PBST, the membranes were incubated with goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) (1:1000, Santa Cruz, Dallas, TX, USA) at room temperature for 1 h. The blots were visualized by enhanced chemiluminescence (ECL) reagents (Thermo Fisher). The images were observed and captured using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). GAPDH (Rabbit anti-GAPDH, 1:100–1:1000, Santa Cruz) was used as an internal reference, and the images were analyzed by ImageJ software [22 (link)].