Figure 3

Elevated protein phosphorylation and AKT kinase activity in human breast cancer lines. Subconfluent proliferating culture of breast cancer lines (MCF-7 and MDA-MB-468), immortalised breast epithelial line (MCF10A), and normal primary HMEC were harvested and lysed. Proteins (100 μg) derived from such lysate were subjected to Western blot analysis using phosphor-specific primary antibodies to detect various phosphorylated proteins (A). In addition, 500 μg of protein from the same lysates were immunoprecipitated with anti-AKT antibody and the resulting immune complexes were further subjected to AKT kinase activity assays using GSK-3α/β as a substrate. The presence of phosphorylated GSK-3α/β (Ser21/Ser9) indicates positive AKT kinase activity in cells (B).