Figure 3.

Analysis of VEGF-DΔNΔC by silver staining, Western blotting, and a bioassay to assess binding to VEGFR-2. (A) Silver stain (Left) and Western blot analysis (Right) of VEGF-DΔNΔC arising from affinity purification (fraction 3). Samples analyzed by silver staining were the fraction containing VEGF-DΔNΔC (+) and a control fraction arising from affinity chromatography of the conditioned medium from cells transfected with expression vector lacking VEGF-D coding sequences (−). Western blot analysis was carried out by using the fraction containing VEGF-DΔNΔC with mAb M2 or a control isotype-matched antibody (Neg). Molecular mass markers (kDa) are indicated. (B) Analysis of VEGF-DΔNΔC using the VEGFR-2 bioassay to assess binding to the extracellular domain of VEGFR-2. Bioassay cells (10⁴ cells) were washed to remove IL-3 and incubated with the recombinant VEGF-DΔNΔC in fraction 3 from the M2 affinity chromatography (VEGF-DΔNΔC). The negative controls were cell culture medium without added growth factor (Medium Alone) and fraction 3 from affinity chromatography of the conditioned medium from cells transfected with expression vector lacking VEGF-D coding sequences (Vector). The positive control was a series of doubling dilutions of mouse VEGF₁₆₄ from an initial concentration of 100 ng/ml (VEGF₁₆₄). VEGF-DΔNΔC was also tested against Ba/F3 cells expressing a chimeric receptor consisting of the extracellular domain of Tie2 and the transmembrane and cytoplasmic domains of EpoR (VEGF-DΔNΔC: Tie2/EpoR). All of the fractions used for the assays were tested at an initial concentration of 10% in cell culture medium followed by doubling dilutions. The concentration of VEGF-DΔNΔC at 10% dilution of fraction 3 was 300 ng/ml. Cells were incubated for 48 h, and cell proliferation was then quantitated by the addition of [³H]thymidine and measuring the amount incorporated over a 4-h period. Assays were carried out in duplicate and error bars denote 1 SD.