Nc membrane

Cells were harvested and lysed with ice-cold lysis buffer (62.5 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10 % glycerol). After centrifugation at 12,000 g for 10 min at 4°C, proteins in the supernatants were quantified and separated by 10 or 12 % SDS-PAGE, either stained by Coomassie Brilliant Blue or transferred to NC membrane (Amersham Bioscience, Buckinghamshire, U.K.). Primary antibodies used as indicated: monoclonal anti-Flag M2 (F1804, Sigma, U.S.A), monoclonal anti-Flag M2-Peroxidase (HRP) antibody (1:5000 dilution, A8592, Sigma, U.S.A), Polyclonal anti-MRFAP1 (1:2000 dilution, 11639-1-AP, Ptglab, Wuhan, Hubei, China), Polyclonal anti-FBXW8 (1:1000 dilution, sc-167864, Santa cruz, U.S.A). Polyclonal anti-Cyclin B1 (1:2000 dilution, 55004-1-Ap, Ptglab, Wuhan, Hubei, China) or polyclonal anti-Cyclin B1(1:1000 dilution, sc-4073, Santa cruz, U.S.A), polyclonal anti-GAPDH (1:5000 dilution, 60004-1-Ig, Ptglab, Wuhan, Hubei, China)

Total proteins were isolated from tissues or cells. Proteins were separated by 10% SDS-PAGE, transferred to NC membrane (Amersham Bioscience, Buckinghamshire, UK). After blocking with 10% nonfat milk for 2 h, membranes were immunoblotted with antibodies overnight, followed by HRP-linked secondary antibodies (Santa Cruz, USA). Protein levels of GAPDH were used as loading controls.

Total proteins were extracted from transfected cells using RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentration of the lysate was quantified using a bicinchoninic acid protein assay (BCA). Equal amounts of proteins were separated on a 7–10% SDS-PAGE gel and subsequently transferred to a methanol-activated NC membrane (Whatman, Dassel, Germany). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature and then incubated overnight with the following antibodies: LEF1 (ab53293, Abcam, 1/5000 dilution), E-cadherin (Cell Signaling Technology, 1/1000 dilution), vimentin (GTX100619, GeneTex, 1/5000 dilution), Snail (GTX100754, GeneTex, 1/500 dilution) and GAPDH (ab181602, Abcam, 1/10,000 dilution). GAPDH was used as a loading control. Then, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (Biosynthesis Biotech, Beijing, China). Next, the membranes were imaged using an ECL Kit (Pierce, Shanghai, China) following the manufacturer’s instructions.

Purified rSjTOR-ed1 protein was subjected to SDS-PAGE and then electroblotted onto a NC membrane (Whatman, Munich, Germany) at 135 mA for 70 min at 4 °C. Membranes were blocked with PBST plus 5% (w/v) nonfat milk for 1 h at 37 °C with constant shaking, and were probed with serum from mice infected with S. japonicum, anti-rSjTOR-ed1 mouse serum, or normal mouse serum diluted 1:100 in PBST overnight at 4 °C. After washing, membranes were probed with goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) diluted 1:3000 in PBST for 1 h at room temperature (RT). Finally, the immunoreactive bands were visualised using DAB Substrate Solution (Tiangen Biotech, Beijing, China) after three washes with PBST.

Cells (1 × 10⁶ cells/well) were seeded in 10-cm dishes in the presence of the MFD. The cells were collected by trypsin-EDTA and lysed in RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1 % NP-40) containing protease inhibitors. After mixing for 30 min at 4 °C, the mixtures were centrifuged (10,000×g) for 10 min at 4 °C and the supernatants were collected as whole-cell extracts. The protein content was determined using the Bio-Rad protein assay reagent and bovine serum albumin as a standard. An equal amount of protein from the total cell extracts was boiled for 8 min. The extracts were separated by SDS-polyacrylamide gels and transferred to a NC membrane (Whatman). The blots were blocked in 5 % non-fat dry milk/PBS for 1 h at room temperature. The blots then were incubated overnight with primary antibodies, followed by horseradish peroxidase-conjugated goat anti-mouse (or rabbit) IgG for 1 h. The immunoreactive bands were revealed by enhanced chemiluminescence with a commercially available ECL kit [18 (link)].