This Mouse HVEM overexpression lysate was created in CHO Stable Cells and intented for use as a Western blot (WB) positive control. Purification of HVEM protein (Cat: 10567-M03S) from the overexpression lysate was verified.
CHO Stable Cells
A DNA sequence encoding the extracellular domain (Met 1-Gln 206) of mouse HVEM (NP_849262.1) precursor was fused with C-terminal His-tagged Fc region of human IgG1 at the C-terminus.
The recombinant mouse HVEM /Fc is a disulfide-linked homodimeric
Protein after removal of the signal peptide. The reduced monomer consists of 415 amino acids and predicts a molecular mass of 46.4 KDa. By SDS-PAGE under reducing conditions, the apparent molecular mass of rmHVEM/Fc monomer is approximately 65 KDa due to the glycosylation.
Mouse HVEM CHO Stable 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.
Herpesvirus entry mediator (HVEM), also referred to as TNFRSF14, TR2 (TNF receptor-like molecule) and ATAR (another TRAF-associated receptor), is a member of type I transmembrane protein belonging to the TNF-receptor superfamily. It is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Two TNF superfamily ligands lymphotoxin α (TNF-β) and LIGHT (TNFSF14) are identified as cellular ligands for HVEM and initiate the positive signaling. However, recent studies have revealed that HVEM is also involved in the unique inhibitory signaling pathway for T cells through activating tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in B and T lymphocyte attenuator (BTLA). HVEM provides a stimulatory signal following engagement with LIGHT (TNFSF14) on T cells. In contrast, it can also provide an inhibitory signal to T cells when it binds the B and T lymphocyte attenuator (BTLA), a ligand member of the Immunoglobulin (Ig) superfamily. Thus, HVEM may be viewed as a molecular switch, capable of facilitating both stimulatory and inhibitory cosignaling in T cells. Substantial evidence from both human disease and from experimental mouse models has indicated that dysregulation of the LIGHT-HVEM-BTLA cosignaling pathway can cause inflammation in the lung and in mucosal tissues.