Zm 323881

hrDkk-3 peptide (R&D Systems, Minneapolis, MN, USA); ML347 (Tocris Bioscience, Bristol, UK); Oligo: SASI_Hs01_00113768 (Sigma Aldrich, Milan, Italy); Recombinant Human TGF beta-1 (Millipore, Billerica, MA, USA); ZM 323881 (Tocris Bioscience).

LEC were stimulated as described above. In some assays, MSC conditioned medium was pre-incubated for 1 h at 37°C with 1 µg/ml sVEGFR-1 or -2 to trap VEGF-A. To specifically inhibit VEGFR-2, LEC and MSC-conditioned medium were pre-incubated with ZM 323881 (Tocris Bioscience, Bristol, UK) at a concentration of 10 nM for 1 h at 37°C. Cells were rinsed with ice-cold PBS and lysed with RIPA buffer containing phosphatase and protease inhibitors (Roche, Mannheim, Germany). Samples were dissolved in SDS buffer and migrated on 10% SDS-PAGE gel before being transferred onto a PVDF membrane. After 1 h blocking in 1% casein, phosphorylated and total proteins were detected by 4°C overnight incubation with the appropriate antibodies, followed by 1 h incubation in HRP (Horseradish peroxidase)-coupled secondary antibody (Cell Signaling, San Diego, CA) and ECL revelation in LAS4000 imager (Fujifilm, Tokyo, Japan). The following antibodies were used: rabbit monoclonal phospho-ERK1/2, ERK1/2, phospho-VEGFR-2 and VEGFR-2 (Cell Signaling, San Diego, CA). For VEGF-A and -C detection a rabbit polyclonal anti VEGF-A antibody (A-20; Santa Cruz Biotechnology, Dallas, TX) and a rabbit polyclonal anti VEGF-C antibody (104-PA10; ReliaTech, Wolfenbüttel, Germany) were used.

Enriched RGC were subjected to different treatment during the whole period of the RGC cultures (6 DIV). Conditioned media were applied at the dilution of 1:2 directly into RGC culture medium. The monoclonal mouse anti-VEGF-A (anti-pan against all VEGF-A isoforms, Santa-Cruz Biotechnology, Dallas, TX, USA), the polyclonal rabbit anti-murine VEGF-A₁₆₄ (Abcam, Cambridge, UK), ranibizumab (Lucentis®, Novartis, Bâle, Switzerland; gift from Pr Matonti), a Fab fragment of the humanized monoclonal VEGF antibody (neutralizes all VEGF isoforms) and a polyclonal Ig-G (rabbit polyclonal anti-NF200; Sigma-Aldrich, Saint-Louis, MO, USA) were incubated directly into the culture medium of RGC cultures. Recombinant rat VEGF-A₁₆₄ protein (R&D systems, Minneapolis, MI, USA), recombinant mouse VEGF-B₁₆₇ protein (R&D systems) and recombinant rat VEGF-D₁₆₄ protein (R&D systems) were also directly added into the medium. The antagonist at VEGF-R2 receptor, ZM 323881 (Tocris Biosciences, Ellisville, MO, USA) was co-incubated with VEGF-A₁₆₄. ZM 323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol) is a potent and selective inhibitor of VEGF-R2 tyrosine kinase in vitro (IC₅₀ < 2 nM), compared with other receptor tyrosine kinases, including VEGF-R1, a range of other receptor tyrosine kinases such as PDGFRβ, FGFR1, EGFR and erbB2 (IC₅₀ > 50 µM)^{45 (link)}.