IGF-1–Induced VEGF and IGFBP-3 Secretion Correlates with Increased HIF-1α Expression and Activity in Retinal Pigment Epithelial Cell Line D407

METHODS. D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 , VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na /K ATPase -1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy.