Akt regulates the expression of key regulators of Stat5 activity. (a) Representative western analysis of expression of proteins that regulate mammary differentiation. Mammary protein lysates from mice with indicated Akt genotypes at day 18.5 of pregnancy. β-Tubulin levels served as a loading control. (b) Quantitative analysis of individual molecule expression in late-pregnant mice (n = 6 mice per genotype). The ratios in each group were normalized to Akt1+/+;Akt2+/+ mice. Statistical analysis was calculated by comparing each group with Akt1+/+;Akt2+/+ mice, except for the indicated P values shown between Akt1-/-;Akt2+/+ mice and Akt1-/-;Akt2+/- mice. *P < 0.05. **P < 0.01. ***P < 0.001. n.s., not significant. (c) Representative western analysis of Stat5a/b activation in Akt1+/+;Akt2+/+ and Akt1-/-;Akt2+/- mammary tissues at day 18.5 of pregnancy treated with prolactin (0.2 μg/ml) for the indicated periods (top panel). Graph shows the ratios of phospho-Stat5 (p-Stat5)/Stat5 at the indicated timepoints calculated from three independent experiments (bottom panel).