Akt1-/-; Akt2+/- mice exhibit impaired secretory epithelial differentiation during late pregnancy and lactation. (a) Northern analysis of milk protein gene expression in mammary glands from pregnant mice (day 18.5) with the indicated Akt genotypes. Cytokeratin 18 (CK18) and 18S rRNA were included as controls for epithelium-specific expression and RNA loading, respectively. WAP, whey acidic protein. (b) Representative western analysis of mammary glands from mice with the indicated Akt genotypes at day 18.5 of pregnancy. Protein lysates were immunoblotted with the indicated antibodies. β-Tubulin levels served as a loading control. (c) Quantitative analysis of Akt/β-tubulin and phospho-S6 (p-S6)/S6 in late-pregnant mice (n = 6 mice per genotype). The ratios were normalized to Akt1+/+;Akt2+/+ mice. Statistical analysis in differential expression 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. n.s., not significant. (d) Immunofluorescence analysis of Na-Pi cotransporter 2B (Npt2b) expression during lactation. Mammary sections from lactating mice with indicated Akt genotypes were immunostained for Npt2b (green). Nuclei were counterstained with Hoechst 33258 (blue). Luminal epithelial cells were counterstained with CK8 (red). Mammary gland tissue from a 6-week-old virgin MTB mouse served as a negative control for staining. Scale bar: 100 μm. (e) Lactose levels of mammary tissues from Akt1+/+;Akt2+/+ mice, Akt1-/-;Akt2+/+ mice, and Akt1-/-;Akt2+/- mice at day 9 of lactation (n = 4 for each genotype).