Natural killer (NK) cells are regulated by stimulatory and inhibitory signals from a variety of receptors. Three main receptor families are responsible for NK cells recognition of MHC I molecules, including Ly-49, CD94/NKG2 and KIR (killer-cell inhibitory receptor). DAP12 is a phosphoprotein that is involved in the activation of NK cells. This protein interacts with membrane glycoproteins of the KIR family, resulting in cellular activation. DAP12 also binds to CD94/NKG2C, an activating NK cell receptor belonging to the C-type lectin superfamily. Additional proteins that bind to DAP12 include Ly-49D and Ly-49H, which associate with DAP12 in the plasma membrane. Phosphorylated DAP12 binds to ZAP-70 and Syk, suggesting that the activation pathway may be similar to that of the T and B cell antigen receptors.
Background References
1. Paloneva J et al. Loss-of-function mutations in TYROBP (DAP12) result in a presenile dementia with bone cysts. Nat Genet 25:357-361 (2000).
2. Kondo T et al. Heterogeneity of presenile dementia with bone cysts (Nasu-Hakola disease): three genetic forms. Neurology 59:1105-1107 (2002).
Sequence Similarity
Belongs to the TYROBP family.
Tissue Specificity
Expressed at low levels in the early development of the hematopoietic system and in the promonocytic stage and at high levels in mature monocytes. Expressed in hematological cells and tissues such as peripheral blood leukocytes and spleen. Also found in bone marrow, lymph nodes, placenta, lung and liver. Expressed at lower levels in different parts of the brain especially in the basal ganglia and corpus callosum.
Post-translational Modification
Following ligand binding by associated receptors, tyrosine phosphorylated in the ITAM domain which leads to activation of additional tyrosine kinases and subsequent cell activation.
Western blot analysis of DAP12 on THP-1 cell lysates with Rabbit anti-DAP12 antibody (ET7108-76) at 1/1,000 dilution.
Lysates/proteins at 20 µg/Lane.
Predicted band size: 12 kDa Observed band size: 12 kDa
Exposure time: 25 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7108-76) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue using anti-DAP12 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7108-76, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Flow cytometric analysis of THP-1 cells labeling DAP12.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET7108-76, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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