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77533
TREM2 Signaling Pathways Sampler Kit
Primary Antibodies

TREM2 Signaling Pathways Sampler Kit #77533

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Confocal immunofluorescent analysis of THP-1 (positive, left) and HL-60 (negative, right) cells using TREM2 (D8I4C) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Immunoprecipitation of DAP12 from THP-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or DAP12 (D7G1X) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using DAP12 (D7G1X) Rabbit mAb.

Flow cytometric analysis of RL-7 cells using Syk (D3Z1E) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Syk(Tyr525/526) (C87C1) Rabbit mAb, or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.

Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.

Western blot analysis of extracts from various cell lines using PLCγ1 (D9H10) XP® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using PLCγ1 (D9H10) XP® Rabbit mAb.

Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or treated with recombinant mouse PDGF-BB (200 ng/ml, 15 min; green), using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Confocal immunofluorescent analysis of HCT 116 cells (left, positive) and 22Rv1 cells (right, negative) using PLCγ2 (E5U4T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Western blot analysis of extracts from untreated or anti-human IgM treated Ramos cells or lamda phosphatase-treated Ramos cell lysates, using Phospho-PLCgamma2 (Tyr759) Antibody (upper) or PLCgamma2 Antibody #3872 (lower).

Immunoprecipitation of TREM2 from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TREM2 (D8I4C) Rabbit mAb. Western blot analysis was performed using TREM2 (D8I4C) Rabbit mAb.

Western blot analysis of extracts from THP-1, U-937, and Raw 264.7 cells using DAP12 (D7G1X) Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Syk (D3Z1E) XP® Rabbit mAb.

Confocal immunofluorescent analysis of Ramos cells, serum-starved (overnight; left) or IgM-treated (12 ug/ml, 2 minutes; right), using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Confocal immunofluorescent analysis of Ramos cells, human IgM treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.

Western blot analysis of extracts from HeLa cells, either mock-transfected or transfected for 48 hours with SignalSilence™ PLCγ1 siRNA I #6293 or siRNA II #6254, using PLCγ1 (D9H10) XP® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human lung carcioma using PLCγ1 (D9H10) XP® Rabbit mAb.

Immunoprecipitation of phospho-PLCγ1 (Tyr783) from A-431 cell extracts stimulated with hEGF #8916 using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb.

Immunoprecipitation of PLCγ2 from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is PLCγ2 (E5U4T) Rabbit mAb. Western blot analysis was performed using PLCγ2 (E5U4T) Rabbit mAb.

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human TREM2 protein (hTREM2-Myc; +), using TREM2 (D8I4C) Rabbit mAb.

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human DAP12 (hDAP12; +), using DAP12 (D7G1X) Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human lymph node using Syk (D3Z1E) XP® Rabbit mAb.

Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM, using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (upper) or Syk Antibody #2712 (lower).

Western blot analysis of extracts from Jurkat cells treated with hydrogen peroxide (2mM for 2 minutes) or with lambda phosphatase and extracts from Ramos cells treated with anti-human IgM (12 micrograms/ml for 2 minutes) using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb.

Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or stimulated with hPDGF-BB #8912 (5 min; +), and from A-431 cells, untreated (-) or stimulated with hEGF #8916 (5 min; +), using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western blot analysis of extracts from various cell lines using PLCγ2 (E5U4T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western blot analysis of extracts from THP-1, HL-60, and Jurkat cells using TREM2 (D8I4C) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Immunohistochemical analysis of paraffin-embedded mouse spleen using Syk (D3Z1E) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Immunoprecipitation of Syk protein from SR cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Syk(D3Z1E) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Syk (D3Z1E) XP® Rabbit mAb

Western blot analysis of extracts from various cell lines using Syk (D3Z1E) XP® Rabbit mAb.

To Purchase # 77533T
Product # Size Price
77533T
1 Kit  (9 x 20 µl) $ 591

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
TREM2 (D8I4C) Rabbit mAb 91068 20 µl
  • WB
  • IP
  • IF
H 28 Rabbit IgG
DAP12 (D7G1X) Rabbit mAb 12492 20 µl
  • WB
  • IP
H M 10, 12 Rabbit IgG
Syk (D3Z1E) XP® Rabbit mAb 13198 20 µl
  • WB
  • IP
  • IHC
  • F
H M R 72 Rabbit IgG
Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb 2710 20 µl
  • WB
  • IP
  • IF
  • F
H 72 Rabbit IgG
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb 2717 20 µl
  • WB
  • IF
  • F
H M 70, 72 Rabbit IgG
PLCγ1 (D9H10) XP® Rabbit mAb 5690 20 µl
  • WB
  • IP
  • IHC
H M R Mk 150 Rabbit IgG
Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb 14008 20 µl
  • WB
  • IP
  • F
H M 155 Rabbit IgG
PLCγ2 (E5U4T) Rabbit mAb 55512 20 µl
  • WB
  • IP
  • IF
H 150 Rabbit IgG
Phospho-PLCγ2 (Tyr759) Antibody 3874 20 µl
  • WB
H M 150 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Specificity / Sensitivity

Each antibody in the TREM2 Signaling Pathways Sampler Kit detects endogenous levels of its human target protein. Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb detects endogenous levels of Syk protein only when phosphorylated at Tyr525/526 of human Syk or Tyr519/520 of mouse Syk. It also detects Syk protein when singly phosphorylated at Tyr526 of human Syk or Tyr520 of mouse Syk. It does not cross-react with other tyrosine-phosphorylated protein tyrosine kinases. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb detects endogenous levels of human and mouse Syk when phosphorylated at Tyr352. It cross-reacts with Zap-70 only when phosphorylated at Tyr319. Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb recognizes endogenous levels of human and mouse PLCγ1 protein only when phosphorylated at Tyr783. Phospho-PLCγ2 (Tyr759) Antibody detects endogenous levels of human and mouse PLCγ2 only when phosphorylated at tyrosine 759. The antibody does not cross-react with phosphorylated PLCγ1.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues around Leu221 of human TREM2 protein, near the carboxy terminus of human Dap12, around Asn463 of human Syk protein, around Leu1220 of human PLCγ1, and near the amino terminus of human PLCγ2. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to Tyr525/526 of human Syk, Tyr352 of human Syk, and Tyr783 of human PLCγ1. P-PLCgamma2 (Y759) Antibody is a polyclonal antibody produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr759 of human PLCγ2 protein. This antibody is purified by protein A and peptide affinity chromatography.

Background

Microglia cells are resident macrophages of the brain that survey the brain environment and dynamically respond to maintain brain homeostasis. Microglial responses include phagocytosis of cellular debris, restricting sites injury or pathology, and/or releasing inflammatory signals to initiate an immune response. Such responses are important during normal development and during diseased states (1).

Recently, the role of microglia in neurodegenerative disease pathology, particularly Alzheimer’s disease (AD), has been of intense investigation. Much of this work is driven by human genetic data that links microglia-enriched genes with AD progression (2). The triggering receptor expressed on myeloid cells 2 (TREM2) protein is an innate immune receptor that is expressed on the cell surface of microglia (3). TREM2 plays a role in innate immunity, and a rare functional variant (R47H) of the TREM2 gene is associated with the late-onset risk of AD (3,4). How TREM2 contributes to disease function is currently an active area of research (4,5), but might drive a number of microglial cellular functions ranging from microgliosis, phagocytosis, and cytokine release via a variety of signaling cascades triggered by TREM2.

The TREM2 receptor is a single-pass type I membrane glycoprotein that consists of an extracellular immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic tail. Ligands for TREM2 include phospholipids, apolipoproteins, and lipoproteins. Upon activation, TREM2 interacts with the tyrosine kinase-binding protein DNAX-activating protein 12 (DAP12, TYROBP) to form a receptor-signaling complex (6). Ligand binding by DAP12-associated receptors, including TREM2, results in phosphorylation of tyrosine residues within the DAP12 immunoreceptor tyrosine-based activation motif (ITAM) by Src family kinases; ITAM phosphorylation leads to activation of spleen tyrosine kinase (Syk) and downstream signaling cascades (7). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain and phosphorylation at these residues (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (8). Syk phosphorylation is also a readout for β-amyloid triggered TREM2 activity (9). Phosphoinositide-specific phospholipase C γ 1/2 (PLCγ1/2) is reported to be down stream of Syk (10). Tyr352 of Syk is involved in the association of PLCγ1 (11); Syk-mediated phosphorylation PLCγ1 at Tyr783 activates PLCγ1 enzymatic activity (12). Interestingly, mutations in the microglia-enriched PLCγ2 gene are associated with AD (13,14,15).

  1. Kierdorf, K. and Prinz, M. (2017) J Clin Invest 127, 3201-9.
  2. McQuade, A. and Blurton-Jones, M. (2019) J Mol Biol.
  3. Colonna, M. (2003) Nat Rev Immunol 3, 445-53.
  4. Yeh, F.L. et al. (2017) Trends Mol Med 23, 512-33.
  5. Ulland, T.K. and Colonna, M. (2018) Nat Rev Neurol 14, 667-75.
  6. Peng, Q. et al. (2010) Sci Signal 3, ra38.
  7. Lanier, L.L. et al. (1998) Nature 391, 703-7.
  8. Zhang, J. et al. (2000) J Biol Chem 275, 35442-7.
  9. Zhao, Y. et al. (2018) Neuron 97, 1023-1031.e7.
  10. Kusuyama, J. et al. (2018) J Cell Physiol 233, 2549-59.
  11. Law, C.L. et al. (1996) Mol Cell Biol 16, 1305-15.
  12. Wang, Z. et al. (1998) Mol Cell Biol 18, 590-7.
  13. Magno, L. et al. (2019) Alzheimers Res Ther 11, 16.
  14. Sims, R. et al. (2017) Nat Genet 49, 1373-84.
  15. Dalmasso, M.C. et al. (2019) Transl Psychiatry 9, 55.

Pathways & Proteins

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For Research Use Only. Not For Use In Diagnostic Procedures.

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