Goat anti rptpβ ζ

For detection of protein-protein interactions, in situ PLA was performed. The components used (Sigma) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe and Detection Reagents Orange. HUVEC or U87MG cells were grown on μ-Chamber 12 well on glass slides (Ibidi©, Martinsried, Germany). After reaching 80% confluence or after appropriate treatment of cells, the assay was performed according to the manufacturer’s instructions. Briefly, after fixation and blocking, cells were incubated with the primary antibodies: mouse anti-VEGF (1:250), rabbit anti-VEGF (1:250), rabbit anti-Flk-1 (1:250), mouse anti-Flk-1 (1:250), mouse anti-NCL (1:50), goat anti-RPTPβ/ζ (1:250) (all from Santa Cruz Biotechnology Inc.), mouse anti-α_νβ₃ (1:500; Merck Millipore), mouse anti-PTN (1:500; Abnova, Heidelberg, Germany) and mouse anti-RPTPβ/ζ (1:250; BD Biosciences). Subsequently, cells were incubated with secondary antibodies conjugated with oligonucleotides. After hybridization and ligation of the oligonucleotides, the DNA was amplified. A detection mixture detected the amplicons, resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized at room temperature with Leica SP5 confocal microscope.

Cells were lysed with RIPA buffer, as previously described [8 (link)]. Three mg of total protein were incubated with primary antibody for 16 h at 4°C under continuous agitation. The primary antibodies used were: mouse anti-VEGF (3 μg), mouse anti-Flk-1 (3 μg), goat anti-RPTPβ/ζ (3 μg), goat anti-PTN (3 μg) (Santa Cruz Biotechnology Inc.) and goat anti-β₃ (1.5 μg; Merck Millipore). Protein A- and protein G-agarose beads (Merck Millipore) were added, samples were further incubated for 2 h at 4°C, and beads with bound proteins were collected by centrifugation (5,000 g for 5 min at 4°C) and washed twice with ice-cold PBS pH 7.4. Immunoprecipitated proteins were resuspended in SDS loading buffer and analyzed by Western blot.