(a) Schematic of the construct used to generate MK-PTEN mice. (b) Southern blot and PCR show transmission of the transgene to the germline. Left: the presence of the 2.1-kb transgene is indicated by the arrow. Right: the 536-bp product corresponds to PTEN. (c) mRNA and (d) protein profile expression of PTEN throughout the development of the mammary gland in MK-PTEN and WT mice. (c) Northern blot analysis was performed using total RNA isolated from mammary gland of WT and MK-PTEN mice at various stages of development. The blot was first hybridized with the full-length hPTEN probe followed by a control 18S probe (data not shown). The ratio of MK-PTEN expression levels to that in WT mice, as normalized to 18S RNA levels, is shown on the graph. Results represent mean ± SD for four MK-PTEN and four WT mice at each stage. (d) Protein extracts from mammary glands of MK-PTEN and WT mice at various stages were subjected to immunoblotting for PTEN. Samples contained equal levels of protein, as confirmed by reprobing each membrane with an anti–α-actin antibody (data not shown). The ratio of PTEN protein level between MK-PTEN and WT mice at various stages of mammary development is represented as the mean ± SD for four MK-PTEN and four WT mice at each stage. V-5 and V-6, virgin 5 and 6 weeks; P-9, P-14, and P-20, pregnancy 9, 14, and 20 days; L-2 and L-10, lactation 2 and 10 days; I-2 and I-6, involution 2 and 6 days.