Smad2 3

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).

Cells were lysed on ice using lysis buffer as instructed before²⁷; then the lysate proteins were collected and further analyzed. Protein A/G agarose or Ni‐NTA beads were used for immunoprecipitation or pulldown assays, respectively. Immunoblotting was performed according to standard method with primary antibodies against pY antibody (Abcam EPR16871), pEGFR (CST#3777S), EGFR (CST#4267), pSTAT3 (CST#4113), STAT3 (CST#9139), CK8 (17514‐1‐AP, Proteintech), CD71 (ABclonal#A5865), Vimentin (10366‐1‐AP, Proteintech), GRB2 (CST#3972), Snail1 (ABclonal#A5243), pIGF‐1R (CST#3021), IGF‐1R (20254‐1‐AP, Proteintech), IRS‐1 (17509‐1‐AP, Proteintech), pVEGFR2 (CST#3817S), VEGFR2 (CST#9698), SHC (10054‐1‐AP, Proteintech), pAKT (CST#4060), pERK1/2 (CST#4370), pJAK1 (CST#3331), AKT (CST#9272), N‐cadherin (22018‐1‐AP, Proteintech), ERK1/2 (CST#4695), JAK1 (CST#3344), LIFR (22779‐1‐AP, Proteintech), His (CST#12698), GP130 (21175‐1‐AP, Proteintech), αSMA (55135‐1‐AP, Proteintech), E‐cadherin (20874‐1‐AP, Proteintech), Smad2/3 (CST#8685), Fibronectin (15613‐1‐AP, Proteintech), FAP (ABclonal#A6349), collagen‐I (14695‐1‐AP, Proteintech), pSmad2/3 (CST#8828), TWIST1 (25465‐1‐AP, Proteintech), and GAPDH (CST#5174) were used at recommended dilutions followed by HRP‐conjugated antibodies and analyzed with the ECL system.