Fig. 1. Expression of Smad4 in adult mouse tissues and conditional knockout Smad4 in mammary glands. (A,B) Northern blot analysis of Smad4 expression in adult tissues. Filters bearing poly A⁺ RNA (A, purchased from Clontech) and total RNA (B) were probed with a probe specific to Smad4 cDNA. Ht, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu, skeletal Muscle; Ki, kidney; Te, testis. (C-G) Immunohistochemical staining using an antibody to Smad4. Primary antibody was not used in sections shown in (D,F) to serve as controls. (C,D) Sections from a wild-type virgin gland. (E,F) Sections from a P14 wild-type gland. (G) A section from an I10 mutant gland, which shows very few cells that are still positive for Smad4 (arrowheads). (H) A Smad4 conditional (Co) and deletion (De) alleles. Deletion of Smad4 exon 8 in the mammary gland is achieved by crossing the Smad4 conditional mice with WAP-Cre transgenic mice. (I) Southern blot showing Cre mediated recombination. DNAs were digested with EcoRV and probed with a 2.2 kb HpaI-EcoRV fragment (black bar). Samples are from mammary glands of Smad4^Co/+ mice (the first lane) and Smad4^Co/CoWAP-Cre (lane 2-6) mice at different stages, including virgin, P16.5, L2, L10 and I10. 9.5 kb, 7.2 kb and 4.3 kb fragments are wild-type (Wt), deleted (De) and conditional (Co) alleles, respectively. (J) Northern blot analysis of RNA isolated from p16 and L10 wild-type (lanes 1 and 3) and mutant (lanes 2 and 4) mice. At least three mutant and control mice at each point were examined. The intensities of bands were measured using software, IP-lab and normalized with loading controls. % expression of mutant relative to control=average intensity of mutant/average intensity of controlx100/100. Scale bar: 60 µm for C-G.

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