PTEN phosphatase activity and phosphorylation state of Akt and MAPK in mammary glands of MK-PTEN and WT mice. (a) Mammary gland protein extracts from MK-PTEN and WT mice (500 μg protein per sample) were subjected to immunoprecipitation with the PTEN polyclonal C20 antibody, and the phosphatase activity was measured as described in the Methods. The phosphatase activity in MK-PTEN and WT is represented as mean ± SD in the graph (n = 4 MK-PTEN and WT mice for each group except in virgin and involution, where n = 2 mice in each group). *P < 0.05, ***P < 0.001. (b) Akt and Erk1/2 activation in mammary gland extracts from MK-PTEN and WT mice at 9 and 14 days of pregnancy. Extracts containing 120 μg of protein were analyzed by immunoblotting with anti-Akt and anti-Erk1/2 antibodies that either recognize the phosphorylated form (pAkt or pErk1/2) or equally recognize the phosphorylated and dephosphorylated forms, i.e., total levels (Akt and Erk1/2). The blots were also analyzed for phospho-FKHR immunoreactivity. These results are representative of at least two identical experiments.