Figure 5

Stat5a/b activation is reduced in the mammary glands of Akt1^-/-; Akt2^+/- mice. (a) Immunofluoresence analysis of phospho-Stat5a/b (p-Stat5) expression in mammary tissues from day 18.5 pregnant mice with the indicated Akt genotypes. Luminal epithelial cells and nuclei were counterstained with anti-CK8 (green) and Hoechst 33258 (blue), respectively. Scale bars represent 50 μm. (b) Quantitative analysis of nuclear p-Stat5 in mammary epithelial cells of late pregnant mice (n = 3 mice per genotype). Statistical analysis in differential activity was calculated by comparing each group with Akt1^+/+;Akt2^+/+ mice, except for the indicated P value shown between Akt1^-/-;Akt2^+/+ mice and Akt1^-/-;Akt2^+/- mice. *P < 0.0001. (c) Representative western analysis of p-Stat5a/b and total Stat5a/b expression in mammary tissues from day 18.5 pregnant mice with indicated Akt genotypes. β-Tubulin levels served as a loading control. (d) Quantitative analysis of p-Stat5/Stat5 in late-pregnant mice (n = 6 mice per genotype). The ratio was normalized to Akt1^+/+;Akt2^+/+ mice. Statistical analysis in differential activity 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 < 0.001. (e) Relative expression of Prlr mRNA in mammary glands from Akt knockout mice at day 18.5 of pregnancy (n = 4 for each genotype). Average expression values normalized to cytokeratin 18 ± standard error of the mean are shown.