Rabbit anti vegf

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.

Protein samples for western blot analysis were prepared as described previously [22] (link). Briefly, MSCs (passages 3–5) were washed three times with ice-cold PBS and then treated with lysis buffer (50 mM Tris-HCl, pH 7.5, containing 2% SDS (Sigma-Aldrich) and a protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were centrifuged for 1 h at 18,000× g at 4°C. The supernatants were collected as whole cell lysates. Protein concentrations were estimated using a DC protein assay (Bio-Rad) with a bovine serum albumin standard. Equal amounts of proteins (10 µg) were resolved by SDS-polyacrylamide gel electrophoresis on 4–20% acrylamide gradient gels (Bio-Rad) and then transferred onto a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA). The membranes were blocked with a blocking reagent (Toyobo, Tokyo, Japan) and then incubated with each primary antibody. The primary antibodies used were: rabbit anti-PCAF, rabbit anti-HIF-1α (Cell Signaling Technology), rabbit anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-β-actin (Cell Signaling Technology). After washing, the membranes were incubated with a peroxidase-labeled secondary antibody (Nichirei, Tokyo, Japan) and visualized using Immunostar LD (Wako). Images were captured digitally using a ChemiDoc XRS+ (Bio-Rad) and analyzed by Image Lab 2.0.1 software (Bio-Rad).