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. 2021 May 19;12:2939. doi: 10.1038/s41467-021-23269-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Left: western blots of P-BAD and total BAD from Bad^+/+ mammary glands at indicated stages (Puberty at 5wk; Adult at 12wk; Pregnant; Involution). Each lane contains lysates from independent mice. Control Bad^3SA mammary gland lysate (first lane) shows specificity of anti-pS112-BAD antibody. Black arrowheads highlight gel shift indicative of BAD phosphorylation. CK18 is used as the loading control for the epithelial compartment. Middle: quantitation of total BAD relative to CK18 loading control, shows increased BAD levels in pregnancy with similar levels in other stages. Right: quantitation of pS112-BAD relative to total BAD shows decreased phosphorylation in adult glands with highest levels in pubertal stages. Data are mean ± SD. For each stage, three independent mice were used. b, c Representative immunofluorescent images from n = 3 independent mice of Bad^+/+ pubertal mammary glands stained for b total BAD and c P-BAD with pS136BAD antibody. The pubertal epithelial tree is composed of bilayer mature ducts capped by multilayer TEBs (inset schematic). Regions representing mature ducts (upper) and TEBs (lower) within the same tissue sections are shown. Total BAD (red), pS136-BAD (red), F-actin (green) and DAPI (blue). BAD localized to the epithelial cells in all experiments. bars = 100 µm. For all p-values, ***P < 0.001, **P < 0.01, *P < 0.05. Statistical test details and exact p-values are provided in Supplementary Data 4. Source data are provided in the Source data file.