Fig. 5. IGFBP-5 and IGFBP-3 inhibit IGF-I and IGF-II-mediated PKB phosphorylation in primary mammary epithelial cells. Cells were serum-starved overnight before treatment with (A) 1-10 nM IGFBP-5±1 nM IGF-I; (B) 1-10 nM IGFBP-3±1 nM IGF-I; and (C) 5 nM IGF-II±10 nM IGFBP-5 or -3. Control cells (CON) were maintained in serum-free medium without treatment. Cell lysates were separated by 10% SDS-PAGE and analyzed by immunoblotting with antibodies specific for phosphorylated PKB (Ser473) or total PKB to indicate equal protein levels. Results are representative of three experiments.