A negative control was run simultaneously by omitting the primary antibody

as human neurons. Lysates were solubilized at 4uC for 1 hr and cleared by centrifugation at 16,000 g for 5 min. Protein concentration was determined by the BCA protein assay kit. In adult nulliparous females, the mammary gland is mostly populated by adipocytes with the embedded epithelial network,. Gestation initiates massive proliferation of the progenitor cells to form lobuloalveolar structures which will ultimately differentiate to milk secreting glandular epithelium upon parturition. Cessation of suckling triggers a drop in lactogenic hormones and heralds the necessity for the involution. The involution process occurs in two stages: In the first stage, despite the abundant alveolar cell death there is no remodeling of the glandular structure, this permits the continuance of secretory function if the suckling is resumed. In the second phase, the superfluous lobuloalveolar cells, their supporting matrix and accumulated milk are cleared by the combined action of lysosomal enzymes and matrix metalloproteinases, and the gland resumes an almost pre-gestation status. Extensive efforts and multiple approaches including gene expression, proteomic profiles and animal knock-out models have identified genes critical to different stages of mammary gland development. Notably, the knock-out models of genes critical for involution have revealed delayed involution but none have actually stopped the process. An undisputable attribute of involution is the significant induction of many proteolytic enzymes, specifically the lysosomal hydrolases. Cathepsins B, D and L are elevated at the reversible stage of involution and YM-155 remain high until 96 hrs postweaning. From functional perspective, this massive surge in activated enzymes is required for the clearance and remodeling of the redundant glandular structures. However, studies in the past decades have uncovered diverse and novel biological functions for these proteases,. Specifically, recent expose of their adipogenic effects depict functional significance far beyond their conventional proteolytic properties. The significance of post-trasnslational modification of genes in developmental processes is just emerging. Studies from our laboratory were among the first to indicate the plasticity of mammary epithelium with respect to Cathepsin D production, post-translational modification and activity. Specifically, at the reversible phase of involution, CatD’s cleavage does not proceed beyond the generation of the single chain active enzyme. This is concomitant with its Tyrosine nitration reported by Zaragoza and colleagues. These precise and timely post-translational modifications prompted us to speculate on CatD’s significance in the involution process and re-population of the mammary tissue with adipocytes. 1 Cathepsin D in Mammary Gland Involution We employed an in vitro approach and treated normal mammary epithelial cells with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19660899 CatD purified from involuting or lactating mouse mammary tissue. This approach exploited the capacity of mammary epithelial cells to capture CatD from the extracellular milieu. Morphological and protein profiling analysis were employed to assess the differential effects of involution-derived CatD. The in vitro approach was further corroborated by an in situ approach using mammary tissue from different developmental stages, and defined a critical and previously unidentified function for CatD in mammary gland involution. Lamin B, nitro-Tyrosine and Acid sphingomyelinase. The HRP-label