Figure 1. Wild type WT1 induces anti-angiogenic VEGF₁₆₅b expression.

A. RT-PCR of mRNA extracted from normal or DDS podocytes using primers that detect both proximal and distal splice isoforms of VEGF. VEGF₁₆₅ and VEGF₁₆₅b cDNAs are used as positive controls. Transfection of DDS podocytes with WT1 (+exon 5/−KTS) restored VEGF₁₆₅b splicing. Boxes indicate exon usage represented by the bands. B. ELISAs for VEGF₁₆₅b or VEGF₁₆₅ protein using a pan-VEGF capture antibody and specific detection antibodies. VEGF₁₆₅ was calculated from the difference between pan-VEGF and VEGF₁₆₅b. C. Immunoblot of protein extracted from normal, DDS podocytes, or DDS cells transfected with wild type WT1(+/−) using antibodies to VEGF₁₆₅b, total VEGF or GAPDH. D. RT-PCR of podocyte cell lines (three replicates) from three different DDS patients with different WT1 mutations show reduced VEGF₁₆₅b expression. E. RT-PCR of mRNA from HeLa and HEK293 cells show expression of VEGF₁₆₅b in HEK293 but not HeLa cells (untr=untransfected), but both cell types have increased VEGF₁₆₅b expression in the presence of WT1 (−/−), and WT1 (+/−). F. Transfection of HEK293 cells with plasmids containing DDS-causing WT1 mutations abolished VEGF₁₆₅b expression. Wild type WT1(+/−) over-expression increases distal splicing relative to proximally spliced isoforms. Bar charts are mean±SEM. See also figure S1.