This Mouse CD73 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD73 protein (Cat: 50231-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse NT5E (NP_035981.1) without the propeptide, corresponding to the amino acid (Met 1-Lys 549) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse NT5E consists of 532 amino acids and has a calculated molecular mass of 59.4 kDa which is also detected in SDS-PAGE under reducing conditions.
Mouse CD73 HEK293 Overexpression Lysate: 使用指南
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.
5'-nucleotidase, also known as NT5E, NTE, and CD73, is a cell membrane protein that belongs to the 5'-nucleotidase family. CD73 is a glycosylphosphatidylinositol (GPI) anchored purine salvage enzyme expressed on the surface of human T and B lymphocytes. CD73 catalyzes the conversion of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to the corresponding nucleosides. CD73 serves as a costimulatory molecule in activating T cells. CD73 generated adenosine functions in cell signaling in many physiologic systems, including intestinal epithelium, ischemic myocardium, and cholinergic synapses. CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and/or function. In CD73-depleted cells, surface levels of the leukocyte adhesion molecules ICAM-1, VCAM-1, and E-selectin increase. CD73 produces extracellular adenosine, which then acts on G protein-coupled purinergic receptors to induce cellular responses. CD73 has also been reported to regulate the expression of pro-inflammatory molecules in mouse endothelium.