Luminescent solution

Forty-eight hours after the transfection of MDA-MB-231 cells, cell lysates were collected using 100 μL of RIPA lysate (Applygen, Beijing, China) and 10 μL of PMSF per well of a six-well plate. The protein concentration of cell lysates was measured with a BCA protein assay kit (Applygen, Beijing, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose (PVDF) (Millipore, Boston, MA, USA) membranes for Western blotting. The membrane was blocked with 5% nonfat dry milk (BD, Franklin, LA, USA) for 2 h, and then diluted with primary antibody caveolin-1 (Affinity, Shanghai, China, 1:1000). β-actin (ZSGB-Bio, Beijing, China, 1:1000) was used as an internal reference. After overnight incubation, the membranes were washed 3 times with TBST (supplemented with 0.1% Tween 20) and incubated with HPR-labeled goat anti-rabbit IgG (ZSGB-Bio, Beijing, China, 1:1000) dilution for 2 h at room temperature. After washing with TBST, it was reacted with a luminescent solution (TransGen Biotech, Beijing, China) and developed under a gel imaging system.

Forty-eight hours after the transfection of MDA-MB-231 cells, cell lysates were collected using 100 μL of RIPA lysate (Applygen, Beijing, China) and 10 μL of PMSF per well of a six-well plate. The protein concentration of cell lysates was measured with a BCA protein assay kit (Applygen, Beijing, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (Millipore, Boston, MA, USA) membranes for western blotting. The membrane was blocked with 5% nonfat dry milk (BD, Franklin, LA, USA) for 2 h, and then diluted with primary antibody YTHDF1 (Abcam, Cambridge, UK, 1:1000), E-cadherin (Affinity, Shanghai, China, 1:1000), N-cadherin(Affinity, Shanghai, China, 1:1000), Vimentin(Affinity, Shanghai, China, 1:1000). β-actin (ZSGB-Bio, Beijing, China, 1:1000) was used as the internal control. After overnight incubation, the membranes were washed three times with TBST (supplemented with 0.1% Tween 20) and incubated with HPR-labeled goat anti-rabbit IgG (ZSGB-Bio, Beijing, China, 1:1000) dilution for 2 h at room temperature. After washing with TBST, it was reacted with a luminescent solution (TransGen Biotech, Beijing, China) and developed under a gel imaging system.