Anti snail1

Antibodies used in our experiments included anti-SHH, anti-Gli1, anti-snail1, and anti-GAPDH antibodies (Affinity Biosciences, China). PVDF membrane, BCA Protein Assay Kit, and BeyoECL Moon kit were acquired from Beyotime Biotechnology, China. Cyclopamine was purchased from Selleck Chemicals, USA. Experimental instruments included AU400 automatic biochemical analyzer (OLYMPUS, Japan), BX51T-PHD-J11 microscope (OLYMPUS Company, Japan), image acquisition system CMOS (OLYMPUS Company, Japan), and Image-Pro Plus (Media Cybernetics Company, USA), etc.

The cells were washed twice with ice-cold PBS and ruptured with RIPA buffer (KeyGEN BioTECH, Nanjing, China), PMSF, and cocktail inhibitor for 30 min at 4°C. The mixture was centrifuged at 12,000 ×g for 10 min at 4°C. Lysate protein concentration was determined using the BCA protein assay kit (ThermoFisher Scientific, USA) in accordance with the manufacturer's protocol. The samples were added with SDS loading buffer and boiled for 10 min, then resolved using 10% SDS-PAGE, and finally, transferred onto polyvinylidene difluoride membranes (Millipore, USA). The blots were blocked with 5% nonfat milk, and the membranes were incubated with appropriate primary antibodies, including anti-PININ (Abcam, #ab108485), anti-METTL3 (Abcam, #ab195352), anti-beta-actin (Affinity, #AF7018), anti-E-cadherin (CST, #14472), anti-Vimentin (Affinity, #AF7013), and anti-snail1 (Affinity, #AF6032) for 1 h at room temperature or overnight at 4°C, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (HRP) and imaging with an electrophoresis gel system (ChemiScope 6000, CLIX, Shanghai, China).