Vegf c

The antibodies used in this study were as follows: EGFR (1:50; # ab52894), HER3 (1:25; # ab5470), HER4 (1:150; # ab19391), VEGFR3 (1:100; # ab27278), VEGF-C (1:50; # ab135506), Wnt5a (1:100; # ab72583), Beta-Catenin (1:100; # ab32572), p-Akt1 (1:100; # ab32505), Akt1 (1:50; #ab59380) were purchased from Abcam company, UK. HER2 (1:100, #4290) was purchased from Cell Signaling Technology, Inc., USA. EphrinA1 (1:100; # sc-911) and EphA3 (1:100; # sc-920) were purchased from Santa Cruz Biotechnology, USA. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako EnVision, USA).

To extract total protein, sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 60 mM Tris-HCl) with protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (GenDEPOT, Katy, TX, USA) was used. WB was performed as previously reported [28 (link)]. The primary antibodies used were as follows: hexokinase 2 (HK2, ab209847, EPR20839, 1:500), glucose transporter 1(GLUT1, ab652, 1:2000), L-type amino acid transporter 1 (LAT1, ab208776, EPR17573, 1:1000), vascular endothelial growth factor B (VEGFB, ab110649, EPR4555, 1:1000), VEGFC (ab135506, 1:2000), CD133 (ab19898, 1:1000) and Snail/Slug (ab180714, 1:1000) from Abcam (Cambridge, UK), signal transducer and activator of transcription 3 (STAT3, #9139, 1:1000), pSTAT3 (#9145, D3A7, 1:2000) from Cell Signaling Technology (Danvers, MA, USA), and β-actin-HRP (sc-47778, C4, 1:5000) from Santa Cruz Biotechnology (Dalla, TX, USA). For the digital visualization of the chemiluminescent WB, the ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) was used. These experiments were replicated at least three times with similar results. ImageJ (National Institutes of Health, Bethesda, MD, USA) and BioRad Image Lab 6 (Bio-Rad) were used for band quantification.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (5 mg/mL; Sigma, St. Louis, MO, USA), dimethyl sulfoxide (DMSO; Sigma). Afatinib (BIBW2992) was purchased from Selleck Chemicals (Houston, TX). All these compounds were dissolved in DMSO at 10 mmol/L as stock solutions and stored at -20°C. Antibodies against GAPDH were from Epitomics (Burlingame, CA). Antibodies against CCR7 (Y59), VEGFR3, VEGFR2, VEGFC, and LYVE-1 were from Abcam (Cambridge, MA, USA). Antibodies against phospho-EGFR (Y1068), EGFR, Akt, phospho-Akt (S473), JAK1/2, phospho-JAK1 (Tyr1022/1023), phospho-JAK2 (Tyr1007/1008), c-Myc, STAT3, phospho-STAT3 (Tyr705), FAK, and phospho-FAK (Tyr925) were purchased from Cell Signaling Technology (Beverly, MA).

Protein was extracted from the hearts using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Duesseldorf, Germany) according to the manufacturer’s instructions. Protein extracts (40 µg) were separated by 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The blots were probed with primary antibodies against VEGFC or VEGFR3 (Abcam, Cambridge, UK). HRP-conjugated anti-rabbit or anti-rat IgG (Southern Biotech, Birmingham, AL) were used as the secondary antibodies.

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibody specific for β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CCL5 and VEGF-C antibodies were purchased from Abcam (Cambridge, MA, USA). Recombinant human VEGF-C was purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human CCL5 and the VEGF-C ELISA kit were purchased from PerpoTech (Rocky Hill, NJ, USA). The miR-507 mimic, miR-507 inhibitor, Lipofectamine 2000 and Trizol were purchased from Life Technologies (Carlsbad, CA, USA). The human miRNome microRNA Profilers QuantiMir™ kit was purchased from SBI (Mountain View, CA, USA). Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (MEM), fetal bovine serum (FBS) and all other cell culture reagents were purchased from Gibco-BRL Life technologies (Grand Island, NY, USA). The dual-luciferase reporter assay kit was purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

Briefly, the protein concentration was determined using the Bradford protein assay kit (Bio-Rad). Then the protein samples were separated by sodium dodecy1 sulphate–polyacrylamide gel electrophoresis, followed by electrophoretically transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibody in TBST at 4℃ overnight. Next, the membrane was washed three times and incubated with the appropriate secondary antibody. The protein bands were visualized using enhanced ECL-associated fluorography. The following antibodies were from Abcam: VEGFC (1:1000 dilution for WB) and ACTB/β-actin (1:100,000 dilution for WB).

Treated and untreated colon cancer cells were centrifuged at 13,000 × g for 10 min at 4°C and the supernatant (20–30 μg of protein) was run on 10% SDS-PAGE gel and transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). The blots were blocked with 5% skim milk, followed by incubation with antibodies against ICAM-1, VCAM-1, VEGFC, CXCR4, TGFBR1, E-cadherin, MMP-2 and MMP-9 were purchased from Abcam (Cambridge, MA, USA); antibodies against SDC2, CDH2 and CCL18 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibody against GAPDH was purchased from Cell Signaling Technology (Danvers, MA, USA). Blots were then incubated with goat anti-mouse or anti-rabbit secondary antibody (Beyotime, Shanghai, China) and visualized using enhanced chemiluminescence (Thermo Scientific).