Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189 - PubMed (original) (raw)

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 VEGF189or 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.