Smad4

Cells were lysed in in RIPA Lysis Buffer (Beyotime Biotechnology, China), and the protein concentration was determined by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for approximately 1.5 h at 90 V-120 V, and then transferred to PVDF membranes (Millipore, USA) at 250 mA for 60 min. After blocked with 5% non-fat milk, the membranes were incubated overnight at 4 °C with specific primary antibodies (FOXM1, 1:200, Santa Cruz Biotechnology, USA; PCDH10, 1:500, Proteintech, China; DACH1, 1:500, Proteintech, China; SMAD4, 1:500, Proteintech, China; β-actin, 1:2000, Proteintech, China). After being washed with TBST (Tris-buffered saline plus Tween-20) three times, the membranes were incubated with corresponding secondary antibodies for 1 h. Bound antibodies were visualized with Li-Cor Odyssey imaging system (Li-COR Biosciences, USA) according to the manufacturer's instructions.

Western blotting was carried out with following primary antibodies: Cited1 (made in our lab or Genetex, GTX114559), Oct4 (made in our lab), Sox2 (made in our lab), phospho-Smad1/5 (Cell Signaling Technology, #9516), phospho-Smad2 (Cell Signaling Technology, #3101), total Smad5 (Cell Signaling Technology, #9517), total Smad2/3 (Cell Signaling Technology, #3102), Smad4 (Proteintech, 10231–1-AP), Bmpr2 (Abcam, ab96826), Elf5 (Santa Cruz, sc-9645), T (R&D, AF2085), Placental lactogen 1 (Santa Cruz, sc-376436), Flag (Abmart, M20018F), β-Actin (HuaBio, M1210–2) and α-Tubulin (Sigma, T5168). The inhibitors were purchased from STEMCELL (SB431542: #72232), Selleckchem (LDN193189: S2618; K02288: S7359; protease inhibitor cocktail: B14002; phosphatases inhibitor: B15002) and PeproTech (Noggin: #120–10 C), respectively.

Protein lysate was obtained from the cultured cells using the RIPA lysis buffer (Solarbio Corporation, Beijing, China). The protein concentration was measured with a bicinchoninic acid assay. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membrane was first incubated with rabbit anti-rat primary antibodies, such as E-cadherin (1:2,000; no. 20874-1-AP), fibronectin (FN; 1:3,000; no. 15613-1-AP), vimentin (1:2,000; no. 10366-1-AP), Smad2 (1:2,000; no. 12570-1-AP), Smad4 (1:2,000; no. 51144-1-AP), zinc-finger-enhancer-binding protein 1 (ZEB; 1:2,000; no. 21544-1-AP) (all from Proteintech Group Inc., Chicago, IL, USA), Smad3 (1:2,000, no. AF6362; Affinity BIO, Scoresby, Australia), Smad7 (1:2,000; no. AF5147; Affinity BIO), and β-actin (1:5,000; no. Ab8226; Abcam, Cambridge, UK), and then incubated with goat anti-rabbit horseradish peroxidase-conjugated-labeled secondary antibodies (1:5,000, # A0208, Beyotime Institute of Biotechnology, Haimen, China) for 1 h at room temperature. The blots were detected using enhanced chemiluminescence.

Cells were lysed, and proteins in the supernatant extracts were quantified using a BCA Protein Assay Kit (Beyotime). Total cell lysates containing 50 µg of protein were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose (NC) membranes (PALL). After blocking with 5% non‐fat dry milk in Tris‐buffered saline‐Tween‐20 (TBST) for 2 hours, the membranes were incubated with primary antibodies [the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody diluted at 1:3000; collagen I, collagen III, lysyl oxidase (LOX), osteopontin (OPN), vimentin, α‐SMA, smad2, p‐smad2, smad3, p‐smad3, smad4, smad7 and p‐smad7 antibodies diluted at 1:2000 or 1:1000] at 4°C overnight. The collagen I, collagen III, p‐smad2, p‐smad3 and p‐smad7 antibodies were purchased from Abcam, and the GAPDH, β‐tubulin, smad2, smad3, smad4, smad7, LOX, OPN, vimentin and α‐SMA antibodies were purchased from Proteintech. After three washes with TBST, the membranes were incubated with the corresponding horseradish peroxidise (HRP)‐conjugated secondary antibody (1:5000, GE, HyClone) at 37°C for 2 hours. The protein bands were visualized with enhanced chemiluminescence (ECL; Advansta) and detected using a ChemiDoc^TM MP imaging system (BIO‐RAD). The protein bands were then scanned using Image Lab^TM Software Version 4.1.