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|Human Cell lysate that Mouse TIMD4 / TIM4 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the extracellular domain of mouse TIMD4 (NP_848874.3) precursor (Met 1-Thr 279) was expressed, with a C-terminal polyhistidine tag.|
|The secreted recombinant mouse TIMD4 comprises 268 amino acids with a predicted molecular mass of 29.3 kDa. As a result of diiferent glycosylation, the apparent molecular mass of rm TIMD4 is approximately 60-70 kDa in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization 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.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
A type I transmembrane protein called TIM4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as TIMD4), which belongs to the immunoglobulin superfamily and TIM family. TIM4 is involved in regulating T-cell proliferation and lymphotoxin signaling. It is a ligand for HAVCR1/TIMD1. Recent reports indicate that dendritic cell (DC)-derived T-cell immunoglobulin and mucin domain molecule (TIM)-4, which is expressed on dendritic cells and macrophages, plays an important role in the initiation of T(H)2 polarization. TIM4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of TIM4 in fibroblasts enhanced their ability to engulf apoptotic cells. TIM4 is phosphatidylserine receptor for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved. Modulation of TIM4 production in dendritic cells (DCs) represents a novel therapeutic approach for the treatment of peanut allergy. The interaction of TIM1/TIM4 played a critical role in sustaining the polarization status of Th2 cells in allergic rhinitis (AR) patients. Cross-linking FcgammaRI by antigen/IgG complexes increased the production of TIM4 by dendritic cells via upregulating tumor necrosis factor-alpha in DCs. Specific immunotherapy (SIT) suppresses the skewed Th2 responses via disrupting the interaction of TIM1/TIM4 in antigen-specific Th2 cells.