Chemiluminescence kit

Total protein extracts were prepared using lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2.5% SDS, 100 mM phenylmethylsulfonyl fluoride (PMSF)) containing protease inhibitor cocktail (Roche). Protein samples were resolved on 4–12% SDS–PAGE gels (Life Technologies) and transferred onto nitrocellulose membranes (Amersham Biosciences/GE Healthcare). Membranes were processed using standard western blot protocols, and signals were detected using a chemiluminescence kit (Santa Cruz Biotechnology). Antibody sources are listed in Supplementary Table 1.

For the Western blot analysis, we mixed 10 ug of protein from each sample with 5 × Laemmli buffer and 5% β-mercaptoethanol and boiled it for 10 min. Briefly, we separated an equal volume of protein by SDS–polyacrylamide gel electrophoresis and transferred it to a nitrocellulose membrane. We then incubated the membranes overnight at 4 °C with a 1:1000 dilution of E-cadherin (clone number H-108, catalog number sc-7870; Santa Cruz Biotechnology, Inc, Dallas, Tx, USA), MMP-7 (catalog number AV46075; Sigma-Aldrich, St. Louis, MO, USA), or a 1:2000 dilution of β-actin antibody (clone number C4, catalog number sc-47778, Santa Cruz Biotechnology) for loading control in a blocking solution. Next, we incubated the membranes with a 1:1000 dilution of the appropriate anti-rabbit (catalog number sc-2004; Santa Cruz Biotechnology) or anti-mouse antibody (catalog number sc-2005; Santa Cruz Biotechnology) in the blocking solution. We viewed blots using the chemiluminescence kit (Santa Cruz Biotechnology), and captured the images with ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).

Protein samples collection was performed using RIPA buffer (KeyGen, Shanghia, China) on ice for 15 min. Total protein contents were quantified by an Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China). Aliquots of protein were loaded onto 10% SDS-PAGE gel, followed by transfer onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were subjected to blocking using 5% bovine serum albumin (BSA) for 1 h. The incubation with primary antibodies was performed at 4 °C overnight: cleaved caspase-3 (1:2000, Abcam, ab2302), Bcl-2 (1:2000; Abcam, ab59348), FZD4 (1:1500; Bioss, bs-13217R), β-catenin (1:1500; Bioss, bs-1165R), non-phospho (Active) β-catenin (1:1000; Cell Signaling Technologies, 8814), cyclin D1 (1:2000; Abcam, ab134175), c-Myc (1:1500; Abcam, ab190026), or GAPDH (1:5000; Bioss, bs-2188R). The next day, the membranes were washed 4 times with TBST buffer and then further labeled with HRP-linked secondary antibody (1:10000; Invitrogen, 61–6520) for 1 h at room temperature. After rinsing for 4 times signal development was conducted using a chemiluminescence kit (Santa Cruz, TX, USA), and the protein bands were photographed under a gel imager (Biorad, CA, USA).

The IFN-α-treated HeLa cells and control cells were collected after 48 h incubation. The cell pellets were lysed with lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, plus proteinase inhibitors. The resolved protein samples by SDS-PAGE were blotted onto Hybond nitrocellular membrane (Amersham Biosciences, Freiburg, Germany). The reaction product was first probed with a primary antibody. After extensively washing, a second antibody conjugated to horseradish peroxidase and specific for the Fc of the first antibody was employed. The reaction products were developed using the chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Total proteins are isolated from rat vascular SMCs treated with 10 µM of PR₂₀ using RIPA lysis buffer. After sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride membranes (PVDF). Membranes were blocked with and incubated overnight at 4°C with the following primary antibodies: Thbs1 (NeoMarkers, MS-421), Egr1 (Cell Signaling, 4154), phospho-FAK(Y397) (Cell Signaling, 3283), FAK (Cell Signaling, 3285), phospho-ERK (Cell Signaling, 4376), ERK (Cell Signaling, 9102), phospho-ERM (Cell Signaling, 3141), ERM (Cell Signaling, 3142), GAPDH (Cell Signaling, 2118). Then, membranes were incubated with second antibodies anti-mouse or anti-rabbit HRP-conjugated antibody (1:1000, Bio-Rad). Blots were visualized using a chemiluminescence kit (Santa Cruz Biotechnology) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Tumor tissues were collected, cut into pieces, and then dissolved in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Invitrogen, United States). Protein concentration was quantified by a BCA Protein Assay Kit (Solarbio, Beijing, China). Ten micrograms of total protein extract was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Separated protein in SDS-PAGE gel was transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, United States). After being blocked with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies overnight at 4°C: anti-IL10, anti-IL17a, anti-CXCL10, anti-IFN-γ, anti-TNFα, anti-TGF-β1, and anti-TNFR2 antibody (Cell Signaling Technology, MA, United States). The membrane was washed three times with TBST for 5 min each. After being washed, the membrane was further incubated with HRP-linked secondary antibody (1:3,000; #7074; Cell Signaling, MA, United States) at room temperature for 1 h. Then the membrane was washed four times with 1 × TBST, and the protein bands were visualized using an enhanced Chemiluminescence Kit (Santa Cruz Biotechnology, Dallas, TX, United States) and photographed on a gel imager system (Bio-Rad, CA, United States).