Fig. 4. IGFBP-3 and IGFBP-5 inhibit IGF-I-mediated IRS-1 phosphorylation in mammary epithelial cells. (A) FSK-7 mammary epithelial cells were serum-starved overnight and treated with either 1 nM IGF-I or a combination of IGF-I and 5 nM IGFBP-5 or IGFBP-3. Control cells (CON) were maintained in serum-free medium without treatment. Immunoprecipitates were separated by 6% SDS-PAGE and analyzed by immunoblotting with a phosphotyrosine antibody or IRS-1 to indicate equal total protein levels. (B) Primary mammary epithelial cells were treated with 1 nM IGF-I±10 nM IGFBP-5 as above. Results are representative of three experiments.