Generation and analysis of Deaf-1 transgenic mice. A) Quantitative RT-PCR analysis of RNA purified from mouse mammary glands at different stages of development (wkV, week-old virgin; dP, days pregnant; dL, days lactation; and dI, days involution) using Deaf-1 and CK18 specific primers. Data are from at least two wild-type mice for each time-point and error bars represent standard deviation from the mean. B) Schematic diagram of the 7 kb transgenic construct showing the MMTV promoter, the HA-Deaf-1 transgene, and the rabbit β-globin plus SV40poly(A) intronic sequences. C) Southern blot analysis of HindIII-digested tail DNA, probed with a [α-32P]-labeled SV40 fragment. Arrow indicates the Deaf-1 fragment. D) Western blot analysis of protein lysates from 8 week-old virgin (8 wkV), 18 days pregnant (18 dP) and 1 day lactating (1 dL) mammary glands from Deaf-29, Deaf-42 and Deaf-1 transgenic strains. Anti-Deaf-1 and anti-HA antibodies were used to detect the HA-tagged Deaf-1 protein. Wild-type mammary gland lysate provided a negative control (upper panel). Samples of each lysate (prior to immunoprecipitation) were subject to immunoblotting with anti-tubulin to control for protein loading (lower panel).