Pb9439

Protein was extracted from the cells were resolved by SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore), and then incubated with primary antibodies diluted in blocking buffer at 4 °C overnight. The following primary antibodies were used: Mouse Anti-β-Actin (HC201, TransGen Biotech, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000), Rabbit Anti EMP3 (DF14661, Affinity, 1/1000), Rabbit Anti TWIST1(AF4009, Affinity, 1/1000), Rabbit Anti SNAI2 (PB9439, Boster, 1/1000), Rabbit Anti FOS (AF5354, Affinity, 1/1000), Mouse Anti-VIM (60330–1-Ig, Proteintech,1/10000), HRP conjugated Goat Anti-Rabbit IgG (H + L) (GB23303, Servicebio, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000)was used. Finally, the antigen–antibody reaction was visualized by the enhanced Pierce ECL Western blotting substrate kit (Thermo Scientific/ Pierce, Rockford, IL, USA).

Radioimmunoprecipitation assay lysis buffer was used for isolation of proteins from cells (Boster, Wuhan, China), while a bicinchoninic acid kit (Boster, Wuhan, China) was used for determination of protein concentration; the protein samples were heated after being combined with a loading buffer (Boster) at a 5 : 1 ratio. The proteins in the gels were translocated in polyvinylidene difluoride membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was incubated with primary antibodies after blocking with 5% nonfit milk powder for 2 h at room temperature (RT). The primary antibodies were used for the analysis after incubating the proteins with the following secondary antibodies: rabbit anti-TOP2A (1 : 2,500; CST, 12286), rabbit anti-E-cadherin (1 : 10,000) (ab40772), rabbit anti-N-cadherin (1 : 10,000) (ab18203), rabbit anti-vimentin (1 : 5,000; Abcam ab92547), rabbit anti-Snail1 (1 : 1,000; Boster, PB0449), rabbit anti-Snail2 (1 : 2,000; Boster, PB9439), rabbit anti-β-actin (1 : 10,000; Boster, BM0627), and rabbit anti-GAPDH (1 : 2,000; Boster, A00227-1). The secondary antibodies (1 : 10,000) were purchased from Proteintech, China. The experiments were repeated three times. Finally, the amplified chemiluminescent system was used to visually detect the signal.