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Abstract

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) is a glycosylated secreted protein. Prior to its secretion, SCUBE3 localizes in the cytoplasm. We observed that SCUBE3 protein trans-localized to the nucleus following Doxorubicin (DOX) treatment. DOX is one of the strongest chemotherapeutic agents and the first-line drug used in breast cancer treatment. SCUBE3 structural analysis showed that it lacks a DNA binding domain. Based on this observation and other preliminary data, we hypothesized that nuclear SCUBE3 protein promotes the survival of cells against Doxorubicin treatment. To investigate this hypothesis, we made a wild-type construct and three constructs with mutated nuclear localization sequences (NLS). These constructs will help to determine whether SCUBE3 requires the predicted NLS to localize to the nucleus in the presence of DOX treatment and study SCUBE3 nuclear function. The constructs were also linked to green fluorescence protein (GFP). GFP is a report protein that will help us monitor the location of SCUBE3 in the cells and determine if putative NLS is required for SCUBE3 translocation. The GFP will also help the differentiation between endogenous and ectopic SCUBE3 proteins. We are also generating stable cells expressing each construct for functional analysis.

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