Anti mettl3

The levels of METTL3, METTL14, AlkBH5, FTO, IL-13, nsp9, N protein, and GAPDH were measured by Western blot analysis. In brief, the treated cells were collected and lysed on ice for 30 min in protein isolation buffer, subjected to SDS-PAGE, and transferred to nitrocellulose membrane. Then, the membrane was blocked with 10% low-fat milk at 37 °C for 4 h and probed with MAb anti-N (1:100; generously offered by Prof. Ping Jiang from the Institute of Nanjing Agricultural University), anti-FLAG (1:2000; Abmart), anti-METTL3 (1:1000; Proteintech), anti-METTL14 (1:1000; Proteintech), anti-AlkBH5 (1:1000; Proteintech), anti-FTO (1:1000; Proteintech), anti-IL-13 (1:1000; Proteintech), or anti-GAPDH (1:10,000; Abcam) at 37 °C for 1 h. After washing, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:1000; Abcam), and the bound proteins were visualized with a chemiluminescence (ECL; Biosharp) reagent.

WB was performed as previously described [26 (link)]. Antibodies used were as follows: anti-KAP1 (1:1000, 15,202–1-AP, Proteintech), anti-YTHDC1 (1:1000, 29,441–1-AP, Proteintech), anti-METTL3 (1:1000, 15,073–1-AP, Proteintech), anti-MYCN (1:1000, 84,406, Cell Signaling Technology), anti-Ub (1:1000, 3936, Cell Signaling Technology), anti-β-actin (1:1000, 3700, Cell Signaling Technology) and anti-GAPDH (1:50000, 60,004–1-Ig, Proteintech).

Immunofluorescence staining was performed at room temperature. Cells were washed in PBS, then fixed with 4% paraformaldehyde for 30 min at room temperature and washed 3 times in PBS. Cells were permeabilized with 0.1% Triton-X100 (Sigma T8787) and washed 3 times in PBS. Then, they were blocked in 5% goat serum in 37 °C for 30 min. Cells were blocked with 5% goat serum and then incubated with the following primary antibodies: anti-METTL3 (1:100, Proteintech, Wuhan, China), anti-FTO (1:100, Proteintech, Wuhan, China), anti-MTNR1B (1:100, abcam, Cambridge, UK). Then, cells were incubated with secondary antibody: goat anti-rabbit IgG H&L Alexa Fluor 488 (1:1000, cell signaling technology, Danvers, MA, USA), for 1 h in the dark. Coverslips were mounted to slides with DAPI. All images were taken using a microscope (Olympus, Tokyo, Japan).

Co-IP was performed by Cloudseq Biotech. The cells were lysed to extract protein by Radio Immunoprecipitation Assay Lysis Buffer (Beyotime) and quantified by an enhanced BCA Protein Assay Kit (Beyotime). Supernatant (20 μl) from the cell lysis was boiled with 5× loading buffer (20 μl) was used as input control for Co-IP. The protein samples were immunoprecipitated with magnetic beads precoated with anti-EIF3A (Huabio) and anti-METTL3 (Proteintech, China) antibody. After that, the proteins binding with beads were eluted and used as the IP group for Western blotting. The protein bands were immunoblotted with specific primary antibodies: rabbit anti-NOP2 (1:1000 dilution, Huabio) and rabbit anti-GAPDH (1:1000 dilution, Huabio). After incubation with goat anti-rabbit secondary antibody (1:1000 dilution, SAB), the membranes were scanned using Bio-rad ChemiDoc MP.

The following antibodies were used: anti-CDK1 (abcam, 2546 ab133327), anti-cyclin D1 (CST, 2922), anti-CDK6 (Abcam, ab151247), anti-cyclin E2 (CST, 4132), anti-PHGDH (Proteintech, 14,719–1-AP), anti-PSPH (Proteintech, 14,513–1-AP), anti-Ki-67 (Abcam, ab15580), anti-SLC1A4 (Proteintech, 13,067–2-AP), anti-p53(CST, 9282), anti-MDM2 (CST, 86,934), anti-hnRNPA2B1 (Proteintech, 14,813–1-AP), anti-Mettl3 (Proteintech, 15,073–1-AP), anti-GAPDH (CST, 2118), anti-Flag (CST, 2368), HRP goat anti-rabbit IgG (Biodragon, BF03008), HRP goat anti-mouse IgG (Biodragon, BF03001), CoraLite488-conjugated goat anti-rabbit IgG (Proteintech, SA00013-2), CoraLite594-conjugated goat anti-mouse IgG (Proteintech, SA00013-3), Nutlin-3 was purchased from Sigma (Shanghai, China).