Ab167655

Total proteins were extracted from tissues and cells. Anti-GAPDH antibody (1:5000, A5441, Sigma), anti-CRIF1 (1:2000, 16260-1-AP, Proteintech), anti-cyclin D1 (1:2000, ab137875, Abcam), anti-cyclin E1 (1:1000, ab52189, Abcam), anti-CDK4 (1:1000, ab137675, Abcam), anti-CDK6 (1:1000, ab151247, Abcam), anti-MMP3 (1:1000, ab52915, Abcam), anti-TGF-β1 (1:500, ab9758, Abcam), anti-TGF-β2 (1:100, ab167655, Abcam), TGF-β receptor 1 (1:1000, ab155258, Abcam), anti-Smad2 (1:1000, 3122, CST), anti-Smad3 (1:1000, 9513, CST), anti-p-Smad2 (1:1000, 3108, CST), anti-p-Smad3 (1:1000, 9520, CST), anti-E-Cadherin (1:2000, ab133597, Abcam), and anti-N-Cadherin (1:1000, ab19348, Abcam) antibodies were used. Immunodetection was performed using an EZ-ECL chemiluminescence detection kit (BeitHaemek, Israel).

Cells were lysed in NP40 buffer on ice, after which the proteins were extracted. The concentration of total protein was measured by the BCA method. Equal amounts of harvested total proteins were loaded onto sodium dodecyl sulfate‐polyacrylamide gels and submitted electrophoresis. Then, the membranes were blocked at room temperature for 2 h and incubated with primary antibodies at 4 °C overnight, followed by HRP‐conjugated secondary antibodies at room temperature for 2 h. After washing three times in TBST, protein bands were observed using an ECL chemiluminescence system (Bio‐Rad). Primary antibodies against N‐cadherin (13116, 1 : 1000; CST, Boston, USA), E‐cadherin (3195, 1 : 1000; CST, Boston, USA), vimentin (5741, 1 : 1000; CST, Boston, USA), snail (3879, 1 : 1000; CST, Boston, USA), GAPDH (MB001, 1 : 1000; Biogot Technology, Nanjing, China), and TGFβ2 (ab167655, 1 : 1000; Abcam, Cambridge, UK) were used.