Figure 1.

Expression of murine VHL increases over the course of normal mammary gland development as epithelial cell content increases. A: Western blotting for VHL was performed using tissue whole cell extracts (WCE, 100 μg/lane) prepared from mid-gestation embryos (15-E) or whole mammary glands harvested from mice over the course of gestation and lactation. Extracts were prepared from mature nulliparous (vir) mice, at day 6, 10 or 15 of pregnancy (6-P, 10-P, 15-P), at mid-lactation (10-L) or 10-L followed by 4 days of forced involution (4-Inv). The same blot was stripped and reprobed with antibodies to cytokeratins 8/18 (CK8/18) to indicate the relative abundance of mammary epithelial cell (MEC) content at each stage of development. Note the increased expression of VHL from day 6 of pregnancy to day 10 of pregnancy that persists throughout lactation, and then dramatically decreases at 4-Inv, a peak period of MEC cell death. The virgin sample was overloaded as indicated by the increased expression of CK8/18 in this lane relative to sample prepared from 6-P mice. B: To confirm efficient deletion of Vhl in response to Wap-Cre-mediated recombination (conditional knockout, CKO), tissue WCE were prepared from whole glands isolated from two independent control (WT) or CKO lactating dams during the third round of lactation. VHL expression is reduced by >90% when normalized for MEC content based on CK8/18 expression.