Fig. 2. IGFBPs are secreted from the basal surface of primary mammary epithelial cells grown on basement membrane. Cells were cultured on reconstituted basement membrane for 6 days in differentiation medium. Medium was replaced with serum-free medium (CON) or serum-free medium containing 2.5 mM EGTA for 10 minutes. Medium was then removed and concentrated before ligand blot analysis to detect IGFBPs and immunoblotting to detect secreted ß-casein. IGFBPs could not be detected in the basement membrane gel in the absence of cells (BM alone). Casein immunoblots and ligand blots are representative of three separate experiments.