Mouse ROBO4 HEK293 Overexpression Lysate: Product Information
This Mouse ROBO4 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of ROBO4 protein (Cat: 51081-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse ROBO4 (NP_083059.2) N-terminal fragment (Met 1-Glu 480) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse ROBO4 (aa 1-480) comprises 454 amino acids and has a calculated molecular mass of 49.6 kDa. As a result of glycosylation, the apparent molecular mass of the recombinant protein is approximately 65 kDa in SDS-PAGE under reducing conditions.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Roundabout homolog 4, also known as magic roundabout and ROBO4 is a member of theimmunoglobulin superfamily and ROBO family. ROBO4 is specifically expressed in endothelial cells. It is expressed at sites of angiogenesis in different tumor types. ROBO4 contains twofibronectin type-III domains and twoIg-like C2-type (immunoglobulin-like) domains. ROBO4 is the fourth identified member of the roundabout receptor family. It is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. ROBO4 is predominantly expressed in embryonic or tumor vascular endothelium and is considered important for vascular development and as a candidate tumor endothelial marker. ROBO4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development. ROBO4 is a receptor for Slit proteins, at least for SLIT2, and seems to be involved in angiogenesis and vascular patterning. ROBO4 may mediate the inhibition of primary endothelial cell migration by Slit proteins. Activating ROBO4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.
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