Figure 1.

Expression of three isoforms of VEGF in human U87MG glioblastoma cells. (a) Reverse transcription PCR of VEGF from total RNA isolated from U87MG cells. cDNA lengths for VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ are 470, 602, and 674 bp, respectively. Equivalent amounts of expression of these three VEGF isoforms could also be detected by RNase-protection assays. (b) VEGF Western blot and ELISA analysis. Lane 1, parental U87MG cells; lanes 2, 3, and 4, VEGF₁₂₁-overexpressing clones 7, 12, and 19, respectively; lanes 5, 6, and 7, VEGF₁₆₅-overexpressing clones 2, 15, and 31, respectively; lanes 8, 9, and 10, VEGF₁₈₉-overexpressing clones 10, 26, and 30, respectively. For each sample, 30 μg of total cell lysate protein was used. The numbers at the bottom are levels of VEGF secretion determined by ELISA analysis in units of ng per 10⁶ cells. CM was collected after the cells were cultured for 48 hr. (c) VEGF Western blot analysis for heparin-enriched VEGF protein from CM of VEGF₁₆₅- and VEGF₁₈₉-overexpressing cells. CM was collected after the cells were cultured for 48 hr in the presence (+) or absence (−) of 100 μg/ml heparin. Designator 1, CM of U87MG cells; designators 2, 3, and 4, CM of VEGF₁₆₅-overexpressing clones 2, 15, and 31; designators 5, 6, and 7, CM of VEGF₁₈₉-overexpressing clones 10, 26, and 30. In b and c: , glycosylated VEGF₁₈₉; •, unglycosylated VEGF₁₈₉ or glycosylated VEGF₁₆₅; ▪, unglycosylated VEGF₁₆₅ or glycosylated VEGF₁₂₁; and ⧫, unglycosylated VEGF₁₂₁. Each of the assays was repeated independently three to six times and with cells of various passage numbers, with similar results.