P jak1

Lysate (Beyotime P0013J) containing protease inhibitor and EDTA was utilized to lyse tissues and cells. Protein was then separated by 10% PAGE gels (Epizyme PG112) and transferred onto PVDF membranes (Millipore IPVH00010). The PVDF membranes were blocked with 10% bovine serum albumin (BSA) and incubated with primary antibodies β-actin (CST 3700S), A1AT (Proteintech 16382-1-AP), c-myc (SANTA sc-40), lamin B1 (CST13435S), p-JAK1 (CST 3331S), JAK1 (CST 3332S), p-STAT3 (CST 9145S), STAT3 (CST 4904S) and CEBPB (Proteintech 23431-1-AP) at 4 °C overnight. Then, the membranes were incubated with secondary antibody (Zsbio zb-2301 and Zsbio zb-2305) at room temperature for 1 h.

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.