Ab195352

Protein solubilization buffer was used for cell lysis. Protein extracts were performed according to the manufacturer’s instructions of Total Protein Extraction Kit (BestBio, Shanghai, China). SDS‒PAGE was using for separating the proteins, after that, proteins were transferred to nitrocellulose membranes (Millipore). Antibodies used in our study were mainly anti-GAPDH (Abways, AB0037), anti-NCBP1(Abcam ab154532, Proteintech 10349-1-AP), anti-METTL3 (Abcam ab195352, BETHYL A301-568A) and anti-MYC (Proteintech 10828-1-AP, Cell Signaling Technology #9402). GAPDH was served as a loading control. Finally, membranes were probed by HRP-conjugated secondary antibody (Servicebio GB23302) and visualized by using chemiluminescent reagents (Millipore, Merck, Darmstadt, Germany).

For immunohistochemical assessment, lymphoid tissue sections from DLBCL patients and controls were subjected to heat-induced epitope retrieval according to standard protocols of a histochemical kit (ZSGB-BIO). Then, primary antibodies anti-NCBP1(Abcam ab154532, Proteintech 10349-1-AP), anti-METTL3(Abcam ab195352, BETHYL A301-568A), and anti-c-MYC (Proteintech 10828-1-AP, Cell Signaling Technology #9402) were applied for a 4 °C overnight incubation. Then, incubation with HRP-conjugated secondary antibodies were performed, following with DAB staining. Finally, the slides were visualized and photographed using a light microscope (Nikon, Ni–U).