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. 2014 Oct 15;28(20):2205–2218. doi: 10.1101/gad.245142.114

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© 2014 Cai et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — E2 up-regulates Wnt4 protein level. (A) Western blot analysis of Wnt4 overexpression (OE) to examine the Wnt4 antibody specificity and knockdown efficiency of two independent Wnt4 shRNA. (B,C) Western blot and quantification analysis of Wnt4 proteins during pregnancy, lactation, and involution. (***) P < 0.01. (D–F) Mammary glands of control and ovariectomized mice (OVX) were harvested and analyzed after 2 wk or 3 wk by Western blot and quantification. Either E2 or Pg was sufficient to enhance Wnt4 protein expression, while the augmentation was more pronounced in the presence of both E2 and Pg. The estrous cycle was not controlled in these experiments. (***) P < 0.01. (G) qPCR analysis of cultured luminal cells indicated that E2 had a subtle influence on the Wnt4 mRNA level, and Pg increased Wnt4 mRNA significantly. (H,I) Western blot analysis of cultured luminal cells indicated that the Wnt4 protein level significantly increased in the presence of E2, Pg, or both. (***) P < 0.01.