Polyvinyl difluoride membrane

Cells were lysed in RIPA lysis buffer containing proteinase inhibitors. Each sample (25 ug) was loaded on a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred onto a polyvinyl difluoride membrane (Bio-Rad, Hercules, CA, USA), and membranes were incubated in blocking buffer containing 5% non-fat dry milk for 1 hour at room temperature. Next, membranes were probed overnight at 4°C with a primary antibody in blocking buffer (1:1000 dilution of anti-phospho-STAT3 and anti-STAT3 mAbs, anti-phospho-P38 and anti-P38MAPK mAbs, anti-phospho-ERK1/2 and anti-ERK1/2 mAbs, anti-phospho-JNK and anti-JNK mAbs) (Cell Signal Technology, Danvers, MA, USA), followed by probing with anti-mouse Ig-HRP conjugates (Amersham Biosciences, Buckinghamshire, UK) in blocking buffer at a dilution of 1:3000 for 2 hours at room temperature. Signals were recorded on HyperFilm MP (Amersham Pharmacia Biotech) and developed in a Kodak X-Omat film developer. Results were scanned and densitometrically analyzed using Scion Image software (Scion Corporation, Frederick, MD, USA).

Insect Sf9 cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, and protease inhibitor cocktail (Cat no.: 539134, Calbiochem, Gibbstown, NJ, USA)). The lysate was then centrifuged at 12,000 xg for 15 minutes at 4°C and the supernatant was collected for use.
Samples of the protein were run on 12% SDS-PAGE homogenous gel and transferred to polyvinyl difluoride membrane (Bio-Rad Laboratories, Richmond, CA). The membrane was blocked in 5% milk at 4°C overnight, incubated for 1 h at room temperature with 1:2000 6xHis mAb with HRP Conjugate (Cat no: 631210, Clontech, Mountain View, CA 94043, USA). It was then visualized using an enhanced chemiluminescence assay (ECL kit, Amersham, London, UK).
To semi-quantitatively determine and compare the protein levels, blot images were analyzed using the ImageJ image analysis software (http://rsbweb.nih.gov/ij/index.html). Here, protein levels were quantified as the mean of integrated optical density.

Cells were lysed in ice cold RIPA buffer with protease inhibitor for 35 min, and centrifuged at 4 °C, 11,000 g for 15 min; 10 µg protein was subjected to SDS-PAGE and subsequently transferred to a polyvinyl difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were incubated with the primary antibody at 37 °C, for 1 h followed by incubation with the secondary antibody at 37 °C for 1 h. The primary antibodies were N-cadherin (Cat. no 22018-1-AP, Proteintech, USA), E-cadherin (Cat. no 60335–1-lg, Proteintech, USA), Vimentin (Cat. no 10366-1-lg, Proteintech, USA) Snail (Cat. no 13099-1-AP, Proteintech, USA), Slug (Cat. no 12129-1-AP, Proteintech, USA). Secondary antibodies were goat anti-mouse HRP (Cat. no SA00001-1, Proteintech, USA) or goat anti-rabbit HRP (Cat. no SA00001-2, Proteintech, USA). β-actin (Cat.no TA-09; Zsbio, China) was used as a housekeeping gene control.

Protein was extracted from IPEC-J2 cells using lysis buffer (Sigma), according to the manufacturer’s instructions. Samples containing equal amounts of protein were run on 10% SDS-polyacrylamide gel and then transferred to a polyvinyldifluoride membrane (Bio-Rad). After blocking with TBST containing 5% nonfat dried milk for 2 h at 37 °C, the membrane was hybridized with the primary antibody of Cell Signaling (TBK1, catalogue no. 3504; IRF3, catalogue no. 4302; p-IRF3, catalogue no. 4947), incubated overnight at 4 °C, and then washed three times with TBST for 10 min each. After washing, the membrane was incubated with secondary antibody for 1 h at 37 °C, then washed three times with TBS/T for 10 min. Clarity western enhanced chemiluminescence (ECL) substrate (Bio-Rad) was used to visualize the signals. The Gel-Pro Analyzer (Media Cybernetics, Inc. Bethesda, MD, USA) was used to quantify protein expression, and the ratio of target proteins’ expression was normalized to β-actin.

HEK293T cells were used for transfection to express various tagged proteins. Cells were subsequently lysed in the lysis buffer (40 mM HEPES pH 7.3, 100 mM NaCl, 0.1 or 1% Triton X-100). After 30 min rotation in a cold room, the lysate was cleared by centrifugation at 17,000 × g for 30 min. Next, the resulting supernatant was incubated with 0.5–1 µg antibody for 2–12 h or 20 µl GFP-Trap agarose beads (ChromoTek) for 2 h in a cold room. After that, the antigen-antibody complex was retrieved using 20 µl Protein A/G beads (Thermo Fisher Scientific). After washing with the lysis buffer, beads were subjected to boiling in 50 µl 2× sodium dodecylsulphate (SDS) sample buffer to elute bound proteins. At last, Western blot was performed to analyze bound proteins according to standard protocol. SDS polyacrylamide gel elecctrophoresis (PAGE) separated proteins were transferred to polyvinyl difluoride membrane (Bio-Rad), which was sequentially incubated with primary and HRP-conjugated secondary antibody. Western blot and molecular weight marker bands were acquired under the chemiluminescence and white-light imaging mode, respectively, using a cooled charge-coupled device of LAS-4000 (GE Healthcare Life Sciences). Molecular weights were manually assigned by aligning the two images. Uncropped blot images are presented in Supplementary Fig. 9.