Yt1454

Total protein was extracted using RIPA buffer (Fdbio science, Hangzhou, China) containing 1% protease and phosphatase inhibitors (Bimake, USA). Protein quantification was performed using BCA Protein Assay Kit (Fdbio science, Hangzhou, China). Protein samples of 40ug from each group were separated by 4-20% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.22 μm PVDF membranes (Millipore, USA). Following a 2-hour blocking step in 3% Bovine Serum Albumin (BSA) at room temperature, the membranes were incubated overnight at 4℃ with primary antibodies including anti‐β-actin (1:5000; YM3028, Immunoway), anti-MMP3 (1:1000, ab52915, Abcam), anti‐E‐cadherin (1:500; YT1454, Immunoway), anti‐ZO-1 (1:500; YN1410, Immunoway), anti-N-cadherin (1:500, YT2988, Immunoway), anti-β-Catenin (1:1000, YM3403, Immunoway), anti-Snail (1:500, YT4351, Immunoway) and anti-Vimentin (1:500, YT4880, Immunoway). The next day, the membranes was washed 3 times with TBST and incubated with HRP-secondary antibody for 2 hours at room temperature. Immunoblots were detected with an imaging system (Bio-Rad, USA) and an enhanced chemiluminescence detection kit (Fdbio science, Hangzhou, China). GAPDH was selected as a loading control.