Western Blot Analysis of Epithelial-Mesenchymal Transition

As our laboratory described previously [6 (link)], the culture medium was discarded and cells were washed with phosphate buffer solution (PBS) three times. RIPA lysis buffer (Beyotime, China) was used to extract protein, and the BCA protein assay kit (Beyotime, China) was used to quantify protein concentration. The protein was separated with 10%–15% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Then, the membranes were immersed in PBST (phosphate buffered solution containing 0.1% Tween-20) with 5% skim milk powder for 1 h at room temperature to block nonspecific antigen. After being washed with PBST, the membranes were incubated with primary antibodies against PFKFB3 (1:1000, Proteintech, USA), vimentin (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:1000, Proteintech, USA), p-smad2/3 (1:1000, Proteintech, USA), smad2/3 (1:1000, Proteintech, USA), TGF-β (1:1000, Proteintech, USA), β-actin (1:1000, Proteintech, USA) for the whole night at 4°C. The membranes were washed with PBST three times the next day and incubated with the secondary antibodies (1:10,000, Proteintech, USA) for 1 h at room temperature. After being washed with PBST three times, the protein expression level in membranes can be detected with an enhanced chemiluminescence detection system (Amersham Imager 600, USA).