Rabbit anti n cadherin

Total proteins were extracted using RIPA buffer (with phenylmethylsulfonyl fluoride at 100:1). After the proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis, they were transferred onto polyvinylidene difluoride membranes. Subsequently, the membranes were incubated with 5% bovine serum albumin (Solarbio, Beijing, China) for 2 h at room temperature (22–25 ℃), followed by incubation with rabbit anti-TAGLN2 antibody (1:1000), rabbit anti-E-cadherin (1:2000), rabbit anti-N-cadherin (1:2000), rabbit anti-vimentin (1:2000), rabbit anti-MMP2 (1:800), and rabbit anti-GAPDH (1:8000) (all purchased from ProteinTech Group Inc., Wuhan, China) at 4 °C for 14–16 h. Thereafter, the collected membranes were washed with TBST (Tris-buffered saline, 0.1% Tween 20) three times. Horseradish peroxidase-labeled goat anti-rabbit antibody (1:2500; ProteinTech Group Inc.) or horseradish peroxidase-labeled goat anti-mouse antibody (1:10,000; Proteintech Group Inc.) was used as a secondary antibody, and after incubation at room temperature (22–25 ℃) for 2 h, the membranes were washed with TBST three times and detected via enhanced chemiluminescence.

Paraffin sections prepared from in vivo experiments were used for immunohistochemistry to detect protein expression levels of E-cadherin, N-cadherin and CCND1. The indirect streptavidin-peroxidase method was utilized based on the manufacturer's instructions. Stained tissue sections were examined separately by two pathologists. The antibodies used were rabbit anti-E-cadherin (1:50, proteintech), rabbit anti- N-cadherin (1:50, proteintech), rabbit anti-CCND1 (1: 250, Abcam) respectively.