Ab103102

Cells were lysed using M-PER Mammalian Protein Extraction Reagent lysis buffer (Thermo Fisher Scientific, United States) and protease inhibitor cocktail (Bimake, China). The BCA protein assay Kit (Epizyme, China) was used to quantitate total protein levels. Proteins were separated by sodium dodecyl sulfate−10% polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinyl difluoride (PVDF) membranes (Millipore, United States). The membranes were blocked for 2 h at room temperature in TBST with 5% non-fat milk and incubated overnight at 4°C with primary antibodies including anti-β-actin (AF7018, 1:3,000, Affinity), anti-DLL3 (ab103102, 1:1,000, Abcam), anti-METTL3 (ab195352, 1:1,000, Abcam), anti-NOTCH3 (5278T, 1:1,000, Cell Signaling Technology), and anti-HES1 (11988S, 1:1,000, Cell Signaling Technology). The membranes were washed three times with TBST and incubated with anti-rabbit IgG HRP-linked antibody (31466, 1:3,000, Thermo Fisher Scientific) in room temperature. Finally, the ChemiDoc XRS + system (Bio-Rad, United States) was used to detect protein signal by Immobilon Western Chemiluminescent HRP substrate (Millipore, United States). The quantifications were evaluated using ImageJ software.

Cells were collected and lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher scientific). The protein was separated on 10% polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. After being blocked, the membranes were incubated at 4°C with the following primary antibodies and followed by a secondary antibody at room temperature. Dilution of primary antibodies and secondary antibodies was as follows: Notch1 (1:500; ab27526; Abcam; Eugene, OR, USA), Notch2 (1:1,000; 5732S; Cell signal Technology; Danvers, MA, USA), Notch3 (1:1,000; 5276; Cell signal Technology), Notch4 (1:5,000; 2423; Cell signal Technology), DLL3 (1:1,000; ab103102; Abcam), Hes1 (1:1,000; D6P2U; Cell signal Technology), GAPDH (1:30,000; AP0063; Bioworld Technology; St Louis, MN, USA), Goat anti-Rabbit IgG-HRP (1:10,000; FDR007; Fdbio science; Hangzhou, Zhejiang, China), and Goat anti-Mouse IgG-HRP (1:5,000; FDM007; Fdbio science). The bands of proteins were confirmed by luminescent visualization using an ECL Western Blotting Detection System (Bio-Rad, Hercules, CA, USA) and quantified using the Quantity One software package (Bio-Rad).