ed secondary antibodies were anti-goat, anti-mouse and anti-rabbit. The reaction products were visualized using the enhanced chemiluminescent kit. For equal loading and quality control of Western blots GAPDH and Lamin B were employed for HMEpCs and Acid sphingomyelinase and Lamin B for mouse mammary tissue extracts. Pepstatin Agarose Chromatography CatD from post nuclear cytosolic fractions of mouse mammary gland at different stages of development was purified to homogeneity by three step purification: pepstatin agarose column chromatography at pH 5.5, followed by DEAE and Sephadex 75 column chromatography using well established 
protocols. The purified products were applied to SDS-PAGE and probed by Western blot analysis. The purified mCatD preparations were diluted in growth medium at 2 mg/ml and added to HMEpC cultures. Purified mCatD was collected from 5 different sets of mice at specific developmental stages noted in the text and used in independent experiments. Experimental Procedure Animals and Ethics Statement Female C57BL mice were used at the following stages of development: lactating, post-lactation/involution. The animals were euthanized with ketamine and xylazine and sacrificed by cervical dislocation under deep anesthesia. Pectoral and inguinal groups of mammary glands were removed, and immediately frozen in liquid nitrogen for later use, or were fixed in 10% neutral buffered formalin, processed in a tissue processor and embedded in paraffin for immunoflourescence analysis. Six sets of mice at each stage of development were employed in these studies. mCatD Treatment of Normal Mammary Epithelial Cells Normal mammary epithelial cells grown either in 16 well chambered coverglass, or on LOXO-101 chemical information Millicell culture inserts and allowed to polarize were treated with purified mCatD from different developmental stages at 2 mg/ml for 47 days. Cultures were monitored for morphological changes, and media was replaced every other day. At the end of culture period, cells were either fixed with ice cold Methanol for phase contrast and/or confocal microscopy or were used to make cytosolic and nuclear extracts. Ethics Statement All the animal protocols were reviewed and approved by Institutional Animal Care and Use Committee of Ann and Robert H. Lurie Children’s Hospital Research Center and Northwestern University Feinberg School of Medicine, which is AALAC accredited. Cell Culture Normal human mammary epithelial cells HMEpC, were maintained in defined mammary epithelial cell medium provided by the company. Cultures were determined to be mycoplasma free using the GeneProbe rapid detection system. Alexa Fluor Labeling of Purified mCatD In some experiments, in order to distinguish the endogenous CatD from the administered mCatD, the latter was labeled with Alexa Fluor 594 using Microscale Protein Labeling Kit and according to the manufactures specification. This labeled product was utilized as described above. Tissue Extraction and Western Blot Analysis The mammary tissues were removed, chilled in liquid nitrogen and were either used immediately or kept frozen at 280uC degrees for later use. The frozen tissue was pulverized in pre-chilled mortar and pestle and homogenized in buffer A. The homogenate was subjected to 3 freeze-thaw cycles, passed several times through a 21-gauge needle and centrifuged to yield a post-nuclear cytosolic fraction which was used for recovery and purification of CatD from different PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19659572 fractions. The pellet was dispersed, washed
