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    R&D Systems human trem 2 antibody
    Endogenous <t>TREM2</t> partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.
    Human Trem 2 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Endogenous TREM2 partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Endogenous TREM2 partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques:

    Upregulation of presenilin 1 (PS1) reduces steady-state levels of TREM2 at the cell surface. ( a ) Following PS1 overexpression, HEK293-TREM2 cells were treated with or without the γ-secretase inhibitor Compound E (CpdE) and subjected to cell surface biotinylation assay. Precipitates from streptavidin-agarose beads were immunoblotted for biotinylated TREM2 and total TREM2 levels (levels of TREM2 in 2% total cell lysates). * P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Upregulation of presenilin 1 (PS1) reduces steady-state levels of TREM2 at the cell surface. ( a ) Following PS1 overexpression, HEK293-TREM2 cells were treated with or without the γ-secretase inhibitor Compound E (CpdE) and subjected to cell surface biotinylation assay. Precipitates from streptavidin-agarose beads were immunoblotted for biotinylated TREM2 and total TREM2 levels (levels of TREM2 in 2% total cell lysates). * P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Over Expression, Cell Surface Biotinylation Assay

    Overexpression of presenilin 1 (PS1) impairs TREM2-mediated phagocytosis in microglial cells. ( a ) PS1 and mCherry were co-transfected into BV2 microglial cells. The cells were then incubated with 6-carboxyfluorescein (FAM)-labeled Aβ42 for 2 h. FAM-Aβ42 uptake was analyzed by fluorescence microscopy. Scale bars, 10 μm. ( b ) Phagocytosis of FAM-Aβ42 in BV2 cells stably expressing PS1 as determined by flow cytometry. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Overexpression of presenilin 1 (PS1) impairs TREM2-mediated phagocytosis in microglial cells. ( a ) PS1 and mCherry were co-transfected into BV2 microglial cells. The cells were then incubated with 6-carboxyfluorescein (FAM)-labeled Aβ42 for 2 h. FAM-Aβ42 uptake was analyzed by fluorescence microscopy. Scale bars, 10 μm. ( b ) Phagocytosis of FAM-Aβ42 in BV2 cells stably expressing PS1 as determined by flow cytometry. * P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Over Expression, Transfection, Incubation, Labeling, Fluorescence, Microscopy, Stable Transfection, Expressing, Flow Cytometry, Cytometry

    Presenilin 1 (PS1) interacts with TREM2. ( a , b ) PS1 constructs were transfected into HEK293 cells stably expressing TREM2 with a Myc-tag at the C terminus (HEK293-TREM2). ( a ) Cell lysates were immunoprecipitated with a Myc antibody or control IgG. Immunoprecipitated proteins were subjected to immunoblotting with an Ab14 antibody to detect full-length PS1 (PS1-FL) and the PS1 N-terminal fragment (NTF), an anti-PS1 loop antibody to detect the PS1 C-terminal fragment (CTF) and a nicastrin (NCT) antibody as indicated. ( b ) Cell lysates were immunoprecipitated with Ab14, anti-PS1 loop or control IgG and immunoblotted with Myc and NCT antibodies. ( c ) Lysates from BV2 microglial cells were immunoprecipitated with Ab14 and immunoblotted with NCT and mouse TREM2 antibodies. ( d ) Vectors expressing wild-type (WT) or mutant PS1 (D385A) were transfected into HEK293-TREM2 cells. Cell lysates were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( e ) Lysates from HEK293-TREM2 cells with or without Compound E (CpdE, a γ-secretase inhibitor) treatment were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( f ) Schematic representations of full-length (1–230) or truncated TREM2 constructs, all tagged with GST at the C terminus. SP, signal peptide; TM, transmembrane domain. ( g ) PS1 was co-expressed with full-length TREM2 or other TREM2 fragments as shown in f in HEK293 cells. Cell lysates were precipitated with Glutathione Sepharose beads and immunoblotted with the PS1 antibody Ab14 or an antibody against GST. PS1 co-precipitation levels were determined by densitometric analysis and normalized with respect to both PS1 expression and precipitated GST. ** P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Presenilin 1 (PS1) interacts with TREM2. ( a , b ) PS1 constructs were transfected into HEK293 cells stably expressing TREM2 with a Myc-tag at the C terminus (HEK293-TREM2). ( a ) Cell lysates were immunoprecipitated with a Myc antibody or control IgG. Immunoprecipitated proteins were subjected to immunoblotting with an Ab14 antibody to detect full-length PS1 (PS1-FL) and the PS1 N-terminal fragment (NTF), an anti-PS1 loop antibody to detect the PS1 C-terminal fragment (CTF) and a nicastrin (NCT) antibody as indicated. ( b ) Cell lysates were immunoprecipitated with Ab14, anti-PS1 loop or control IgG and immunoblotted with Myc and NCT antibodies. ( c ) Lysates from BV2 microglial cells were immunoprecipitated with Ab14 and immunoblotted with NCT and mouse TREM2 antibodies. ( d ) Vectors expressing wild-type (WT) or mutant PS1 (D385A) were transfected into HEK293-TREM2 cells. Cell lysates were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( e ) Lysates from HEK293-TREM2 cells with or without Compound E (CpdE, a γ-secretase inhibitor) treatment were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( f ) Schematic representations of full-length (1–230) or truncated TREM2 constructs, all tagged with GST at the C terminus. SP, signal peptide; TM, transmembrane domain. ( g ) PS1 was co-expressed with full-length TREM2 or other TREM2 fragments as shown in f in HEK293 cells. Cell lysates were precipitated with Glutathione Sepharose beads and immunoblotted with the PS1 antibody Ab14 or an antibody against GST. PS1 co-precipitation levels were determined by densitometric analysis and normalized with respect to both PS1 expression and precipitated GST. ** P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Construct, Transfection, Stable Transfection, Expressing, Immunoprecipitation, Mutagenesis

    Mutations in TREM2 affect colocalization and interactions between TREM2 and presenilin 1 (PS1). PS1 was transfected into HEK293 cells stably expressing Myc-tagged TREM2 WT or TREM2 mutants as indicated. ( a , b ) Cells were then subjected to immunostaining with antibodies against Myc, PS1, and TGN46 (a marker for the Golgi, a ) or PDI (a marker for the ER, b ). White arrows in magnified images indicate colocalizing overlap for TREM2, PS1 and TGN46/PDI. Scale bars for a , b , 10 μm. ( c ) Quantification of colocalized signals. Pearson’s correlation coefficient is shown. *** P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Mutations in TREM2 affect colocalization and interactions between TREM2 and presenilin 1 (PS1). PS1 was transfected into HEK293 cells stably expressing Myc-tagged TREM2 WT or TREM2 mutants as indicated. ( a , b ) Cells were then subjected to immunostaining with antibodies against Myc, PS1, and TGN46 (a marker for the Golgi, a ) or PDI (a marker for the ER, b ). White arrows in magnified images indicate colocalizing overlap for TREM2, PS1 and TGN46/PDI. Scale bars for a , b , 10 μm. ( c ) Quantification of colocalized signals. Pearson’s correlation coefficient is shown. *** P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Transfection, Stable Transfection, Expressing, Immunostaining, Marker

    Knock‐down of ADAM 10 is less effective at reducing the shedding of H157Y TREM 2 Western blot of HEK293 cells for ADAM10 (A) or ADAM17 and reprobed for β‐actin (B) confirmed siRNA‐mediated knock‐down of target proteins (arrowheads). siRNA pools, or vehicle, added to cells as indicated. β‐actin (asterisk) was the loading control. Quantitation of WT and H157Y (SEM error bars) TREM2 NTF in the conditioned media of HEK293 cells as compared to untreated cells measured by MSD assay ( N = 8): ADAM10 siRNA reduced shedding whether applied alone or in combination with ADAM17 siRNA. ADAM17 siRNA was ineffective. Fractional inhibition of shedding was greatest for WT TREM2 using ADAM10 siRNA. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Knock‐down of ADAM 10 is less effective at reducing the shedding of H157Y TREM 2 Western blot of HEK293 cells for ADAM10 (A) or ADAM17 and reprobed for β‐actin (B) confirmed siRNA‐mediated knock‐down of target proteins (arrowheads). siRNA pools, or vehicle, added to cells as indicated. β‐actin (asterisk) was the loading control. Quantitation of WT and H157Y (SEM error bars) TREM2 NTF in the conditioned media of HEK293 cells as compared to untreated cells measured by MSD assay ( N = 8): ADAM10 siRNA reduced shedding whether applied alone or in combination with ADAM17 siRNA. ADAM17 siRNA was ineffective. Fractional inhibition of shedding was greatest for WT TREM2 using ADAM10 siRNA. Source data are available online for this figure.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Western Blot, Quantitation Assay, Inhibition

    The disease‐linked H157Y variant of TREM 2 is shed more rapidly than wild‐type TREM 2 Western blot for TREM2 in lysates of HEK293 cells transiently expressing either wild‐type (WT) or the H157Y variant protein ( N = 3): levels of immature TREM2 (major band at 35 kDa) were unchanged by the H157Y substitution; however, total levels of the variant were reduced as compared to WT because of a more marked reduction in the levels of the glycosylated isoform. The proteolytic cleavage of TREM2 generated a truncated C‐terminal fragment (CTF) that was more abundant in lysates from cells expressing H157Y TREM2. GAPDH was the loading control; DAP12 was co‐expressed with TREM2. Molecular mass markers in kDa. Quantitation of the full‐length TREM2 isoforms as shown in panel (A) (data plotted as mean ± SEM; N = 12). Western blot for the shed TREM2 NTF from the conditioned medium of HEK293 cell cultures ( N = 3): levels of H157Y TREM2 NTF were higher than WT. A secreted fragment of the amyloid precursor protein (sAPPa) was the loading control. Molecular mass markers in kDa. The proteolytic fragments of TREM2 as shown in panel (A) (CTF, N = 3) and panel (C) (NTF, N = 15) were corrected for the total full‐length TREM2 (FL) from each cell lysate: the levels of the shed N‐terminal fragment (NTF) of TREM2 were higher in cells expressing the H157Y variant as compared to WT. Data plotted as mean ± SEM. Western blot for TREM2 from the conditioned medium of HEK293 cell cultures treated with varying concentrations of either GI254023X or batimastat (bat): inhibition of TREM2 NTF shedding by GI254023X was equivalent for both variant and WT TREM2; however, more shedding was observed at the higher concentration of batimastat for H157Y TREM2 as compared to WT. Molecular mass markers in kDa. Quantification of total TREM2 shed from HEK293 cells as shown in panel (E) ( N = 7). Maximal concentrations of GI254023X blocked shedding equally for WT and variant TREM2. Batimastat‐resistant shedding was more marked for H157Y TREM2. Data plotted as mean ± SEM. Schematic showing the proteolytic enzymes expected to be active in the presence of the protease inhibitors used in (E and F). MMP: matrix metalloproteinase. Data information: Concentration of inhibitors in micromolar. Two‐tailed Student's t ‐test, P ‐values: * P

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: The disease‐linked H157Y variant of TREM 2 is shed more rapidly than wild‐type TREM 2 Western blot for TREM2 in lysates of HEK293 cells transiently expressing either wild‐type (WT) or the H157Y variant protein ( N = 3): levels of immature TREM2 (major band at 35 kDa) were unchanged by the H157Y substitution; however, total levels of the variant were reduced as compared to WT because of a more marked reduction in the levels of the glycosylated isoform. The proteolytic cleavage of TREM2 generated a truncated C‐terminal fragment (CTF) that was more abundant in lysates from cells expressing H157Y TREM2. GAPDH was the loading control; DAP12 was co‐expressed with TREM2. Molecular mass markers in kDa. Quantitation of the full‐length TREM2 isoforms as shown in panel (A) (data plotted as mean ± SEM; N = 12). Western blot for the shed TREM2 NTF from the conditioned medium of HEK293 cell cultures ( N = 3): levels of H157Y TREM2 NTF were higher than WT. A secreted fragment of the amyloid precursor protein (sAPPa) was the loading control. Molecular mass markers in kDa. The proteolytic fragments of TREM2 as shown in panel (A) (CTF, N = 3) and panel (C) (NTF, N = 15) were corrected for the total full‐length TREM2 (FL) from each cell lysate: the levels of the shed N‐terminal fragment (NTF) of TREM2 were higher in cells expressing the H157Y variant as compared to WT. Data plotted as mean ± SEM. Western blot for TREM2 from the conditioned medium of HEK293 cell cultures treated with varying concentrations of either GI254023X or batimastat (bat): inhibition of TREM2 NTF shedding by GI254023X was equivalent for both variant and WT TREM2; however, more shedding was observed at the higher concentration of batimastat for H157Y TREM2 as compared to WT. Molecular mass markers in kDa. Quantification of total TREM2 shed from HEK293 cells as shown in panel (E) ( N = 7). Maximal concentrations of GI254023X blocked shedding equally for WT and variant TREM2. Batimastat‐resistant shedding was more marked for H157Y TREM2. Data plotted as mean ± SEM. Schematic showing the proteolytic enzymes expected to be active in the presence of the protease inhibitors used in (E and F). MMP: matrix metalloproteinase. Data information: Concentration of inhibitors in micromolar. Two‐tailed Student's t ‐test, P ‐values: * P

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Variant Assay, Western Blot, Expressing, Generated, Quantitation Assay, Inhibition, Concentration Assay, Two Tailed Test

    Titration of batimastat against WT and H157Y TREM 2 shedding The concentration of batimastat in the culture medium of HEK293 cells expressing either WT or H157Y TREM2 (with hDAP12) was varied over 4.5 orders of magnitude and shed TREM2 quantified bt MSD assay 24 h later. More batimastat‐resistant shedding of the variant TREM2 is apparent at higher inhibitor concentrations as compared to the WT protein. Data plotted as mean ± SEM.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Titration of batimastat against WT and H157Y TREM 2 shedding The concentration of batimastat in the culture medium of HEK293 cells expressing either WT or H157Y TREM2 (with hDAP12) was varied over 4.5 orders of magnitude and shed TREM2 quantified bt MSD assay 24 h later. More batimastat‐resistant shedding of the variant TREM2 is apparent at higher inhibitor concentrations as compared to the WT protein. Data plotted as mean ± SEM.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Titration, Concentration Assay, Expressing, Variant Assay

    The disease‐linked H157Y variant of TREM 2 is shed more rapidly from the cell surface (A) Western blot of HEK293‐HaloTag‐hTREM2 cells demonstrating full‐length protein (FL) and also levels of the TREM2 C‐terminal fragment (CTF) that were higher for the H157Y variant (H157Y) as compared to wild type, particularly at 24 h (quantified in C). (B) Likewise, the NTF from the same cells accumulated more in the H157Y variant‐expressing cell supernatants (quantified in D). (E) Levels of DAP12, expressed from the same plasmid, were unchanged. Inhibition of shedding by GI254023X was equivalent for both isoforms of TREM2 (F); however, the metalloprotease inhibitor batimastat (G) was less effective at blocking shedding of the H157Y variant. Data information: Data plotted as mean ± SEM. Two‐tailed Student's t ‐test. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: The disease‐linked H157Y variant of TREM 2 is shed more rapidly from the cell surface (A) Western blot of HEK293‐HaloTag‐hTREM2 cells demonstrating full‐length protein (FL) and also levels of the TREM2 C‐terminal fragment (CTF) that were higher for the H157Y variant (H157Y) as compared to wild type, particularly at 24 h (quantified in C). (B) Likewise, the NTF from the same cells accumulated more in the H157Y variant‐expressing cell supernatants (quantified in D). (E) Levels of DAP12, expressed from the same plasmid, were unchanged. Inhibition of shedding by GI254023X was equivalent for both isoforms of TREM2 (F); however, the metalloprotease inhibitor batimastat (G) was less effective at blocking shedding of the H157Y variant. Data information: Data plotted as mean ± SEM. Two‐tailed Student's t ‐test. Source data are available online for this figure.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Variant Assay, Western Blot, Expressing, Plasmid Preparation, Inhibition, Blocking Assay, Two Tailed Test

    Validation data for the shed TREM 2 MSD assay The MSD assay detected TREM2 in the conditioned media (A) and lysates (B) of HEK293 cells stably expressing hTREM2 and of primary human macrophages but not in parental HEK293 cultures. ND = not detected.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Validation data for the shed TREM 2 MSD assay The MSD assay detected TREM2 in the conditioned media (A) and lysates (B) of HEK293 cells stably expressing hTREM2 and of primary human macrophages but not in parental HEK293 cultures. ND = not detected.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Stable Transfection, Expressing

    Surface biotinylation and fate of TREM 2 Surface‐expressed TREM2 on human macrophages was biotinylated at t = 0 and fractionated into four pools: supernatant, membrane‐associated, cytosolic and nuclear. Similar fractionation was undertaken following incubation for 0.5, 1.0 and 4.5 h. TREM2‐associated biotin was purified by immunoprecipitation and quantified by MSD for biotinylated TREM2. The raw and processed data are presented here. The left block of data shows biotin TREM2 measurements for four timepoints for four biological replicates for each of the four subcellular pools. The right block of data represents the normalised TREM2 quantification, with the membrane fraction at t = 0 defined as 100%.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Surface biotinylation and fate of TREM 2 Surface‐expressed TREM2 on human macrophages was biotinylated at t = 0 and fractionated into four pools: supernatant, membrane‐associated, cytosolic and nuclear. Similar fractionation was undertaken following incubation for 0.5, 1.0 and 4.5 h. TREM2‐associated biotin was purified by immunoprecipitation and quantified by MSD for biotinylated TREM2. The raw and processed data are presented here. The left block of data shows biotin TREM2 measurements for four timepoints for four biological replicates for each of the four subcellular pools. The right block of data represents the normalised TREM2 quantification, with the membrane fraction at t = 0 defined as 100%.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Fractionation, Incubation, Purification, Immunoprecipitation, Blocking Assay

    Shedding of glycosylated TREM 2 NTF is sensitive to inhibitors of ADAM 10 and matrix metalloproteinases Western blotting of the conditioned media from primary murine microglia (left panel), transfected HEK293 cells (middle panel) and primary human macrophages (right panel) revealed the presence of shed wild‐type TREM2. This soluble TREM2 appeared as a > 35 kDa smear of various glycoforms and upon deglycosylation was reduced to a single band of 17 kDa (arrowhead). The ADAM10 inhibitor, GI254023X, blocked shedding in transfected HEK293 cell and macrophage cultures. The concentration of shed TREM2 in the supernatant of macrophage cultures was reduced to ˜50% by 20 μM of both GI254023X and the broad‐spectrum metalloprotease inhibitor GM6001, as measured by an MSD assay. By contrast, the broad‐spectrum serine protease inhibitor PMSF did not reduce shedding. Experiments were repeated three times and for two donors of the macrophage progenitors. Data plotted as mean ± SEM. In HEK293 cells, GI254023X and GM6001 had comparable potencies; however, the MMP2/9 inhibitor SB‐3CT did not block shedding. Data plotted as mean ± SEM; n = 3 replicates. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Shedding of glycosylated TREM 2 NTF is sensitive to inhibitors of ADAM 10 and matrix metalloproteinases Western blotting of the conditioned media from primary murine microglia (left panel), transfected HEK293 cells (middle panel) and primary human macrophages (right panel) revealed the presence of shed wild‐type TREM2. This soluble TREM2 appeared as a > 35 kDa smear of various glycoforms and upon deglycosylation was reduced to a single band of 17 kDa (arrowhead). The ADAM10 inhibitor, GI254023X, blocked shedding in transfected HEK293 cell and macrophage cultures. The concentration of shed TREM2 in the supernatant of macrophage cultures was reduced to ˜50% by 20 μM of both GI254023X and the broad‐spectrum metalloprotease inhibitor GM6001, as measured by an MSD assay. By contrast, the broad‐spectrum serine protease inhibitor PMSF did not reduce shedding. Experiments were repeated three times and for two donors of the macrophage progenitors. Data plotted as mean ± SEM. In HEK293 cells, GI254023X and GM6001 had comparable potencies; however, the MMP2/9 inhibitor SB‐3CT did not block shedding. Data plotted as mean ± SEM; n = 3 replicates. Source data are available online for this figure.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Western Blot, Transfection, Concentration Assay, Protease Inhibitor, Blocking Assay

    TREM 2 expression, glycosylation and proteolysis Surface TREM2 was detected on non‐permeabilised primary human macrophages labelled with anti‐TREM2 polyclonal antiserum (1, red), but not a control antiserum (2) and by live cell immunostaining of HEK293 stably transfected with wild‐type hTREM2 (3, pink; nuclei stained with Hoechst) but not on parental HEK293 (4). Surface immunolocalisation was also observed. Scale bars = 20 μm. Western blots of lysates (L) and supernatants (S) for hTREM2 from parental HEK293 vs. HEK293+hTREM2 cells showed distinct isoforms of TREM2. The cell lysate (HEK293+hTREM2, L) yielded an immature glycoform at 35 kDa with a less intense smear up to 50 kDa. This smear was the predominant species in the supernatant (HEK293+hTREM2, S). Similar distributions of TREM2 were seen in primary human macrophages (Macrophage, L and S). Subcellular fractionation of macrophages over a time course revealed the fate of surface‐biotinylated TREM2 (membrane‐associated in blue circles), indicating that most protein was shed into the supernatant (red squares), with a half time of

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: TREM 2 expression, glycosylation and proteolysis Surface TREM2 was detected on non‐permeabilised primary human macrophages labelled with anti‐TREM2 polyclonal antiserum (1, red), but not a control antiserum (2) and by live cell immunostaining of HEK293 stably transfected with wild‐type hTREM2 (3, pink; nuclei stained with Hoechst) but not on parental HEK293 (4). Surface immunolocalisation was also observed. Scale bars = 20 μm. Western blots of lysates (L) and supernatants (S) for hTREM2 from parental HEK293 vs. HEK293+hTREM2 cells showed distinct isoforms of TREM2. The cell lysate (HEK293+hTREM2, L) yielded an immature glycoform at 35 kDa with a less intense smear up to 50 kDa. This smear was the predominant species in the supernatant (HEK293+hTREM2, S). Similar distributions of TREM2 were seen in primary human macrophages (Macrophage, L and S). Subcellular fractionation of macrophages over a time course revealed the fate of surface‐biotinylated TREM2 (membrane‐associated in blue circles), indicating that most protein was shed into the supernatant (red squares), with a half time of

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Expressing, Immunostaining, Stable Transfection, Transfection, Staining, Western Blot, Fractionation

    Peptidomimetic protease inhibitors point to residues 158–160 as the site of sheddase cleavage An overlapping library of retro‐inverso peptides were designed to mimic the extracellular peri‐membranous domain of TREM2 in the region of the sheddase site (TM: transmembrane). Blue boxes represent retro‐inverso peptides that reduce TREM2 shedding; red boxes those that do not. The black box indicates the three residues that are common to all the inhibitory retro‐inverso peptides. The peptidomimetics were incubated with primary human macrophages, and the resulting levels of shed TREM2 NTF were quantified by MSD ELISA. Blue = inhibitory; red = non‐inhibitory. Values plotted: mean ± SEM; each experiment was repeated for 3–5 independent human donors. Peptides including amino acids 158–160 (blue) inhibited TREM2 shedding more than retro‐inverso peptides that did not (red). Values plotted: mean ± SEM; two‐tailed Student's t ‐test, *** P = 0.003; each experiment was repeated for 3–5 independent human donors. Forward and reverse TREM2 peptidomimetics containing residues 158–160 suppressed TREM2 shedding equally. Values plotted: mean ± SEM; n = 3 replicates; two‐tailed Student's t ‐test; ns = not significant.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Peptidomimetic protease inhibitors point to residues 158–160 as the site of sheddase cleavage An overlapping library of retro‐inverso peptides were designed to mimic the extracellular peri‐membranous domain of TREM2 in the region of the sheddase site (TM: transmembrane). Blue boxes represent retro‐inverso peptides that reduce TREM2 shedding; red boxes those that do not. The black box indicates the three residues that are common to all the inhibitory retro‐inverso peptides. The peptidomimetics were incubated with primary human macrophages, and the resulting levels of shed TREM2 NTF were quantified by MSD ELISA. Blue = inhibitory; red = non‐inhibitory. Values plotted: mean ± SEM; each experiment was repeated for 3–5 independent human donors. Peptides including amino acids 158–160 (blue) inhibited TREM2 shedding more than retro‐inverso peptides that did not (red). Values plotted: mean ± SEM; two‐tailed Student's t ‐test, *** P = 0.003; each experiment was repeated for 3–5 independent human donors. Forward and reverse TREM2 peptidomimetics containing residues 158–160 suppressed TREM2 shedding equally. Values plotted: mean ± SEM; n = 3 replicates; two‐tailed Student's t ‐test; ns = not significant.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Mass spectrometry identifies His157‐Ser158 as the sheddase site TREM2 was immunoprecipitated and deglycosylated from primary human macrophage conditioned media. Two specific bands were visible on a silver‐stained SDS–PAGE gel (arrowheads 1 2). These bands were excised, digested with trypsin and analysed by LC‐MS/MS. TREM2 in the conditioned media of primary human macrophages (black), primary murine microglia (white) and HEK293 cells stably expressing hTREM2 (grey) was digested with trypsin and the resulting peptides identified by mass spectrometry. The most frequent C‐terminal residue not consistent with trypsin digestion was H157 (from two donors/biological replicates of HEK293 cells; each replicate assayed in separate mass spectrometry laboratories; n = total number of peptides identified; where peptide sequences differ between species they are shown as human/mouse). Trypsin digestion (red sites) of band 1 provided almost complete coverage of macrophage TREM2 (bold), lacking only the peptide expected to have R52 at its C‐terminus. Non‐trypsin cleavage (blue) was observed predominantly at H157. The absence of the peptide with K42 at its C‐terminus suggests that N‐terminal truncation is responsible for generating band 2. Underlined: predicted secretion signal peptide. Peptides from the supernatants of HEK293 cells transiently expressing wild‐type (black) and H157Y (white) human TREM2, and co‐expressing human DAP12, were identified by mass spectrometry. The most common C‐terminus was residue 157 for both TREM2 isoforms (one biological replicate; n = total number of peptides identified; where WT and variant sequences vary they are shown as WT/variant). Schematic of the TREM2 protein. SP, signal peptide; IG domain, immunoglobulin domain; TM, transmembrane domain; triangles, N ‐glycosylation sites; arrow, site of proteolytic shedding; all numbers relate to amino acid positions. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

    doi: 10.15252/emmm.201707673

    Figure Lengend Snippet: Mass spectrometry identifies His157‐Ser158 as the sheddase site TREM2 was immunoprecipitated and deglycosylated from primary human macrophage conditioned media. Two specific bands were visible on a silver‐stained SDS–PAGE gel (arrowheads 1 2). These bands were excised, digested with trypsin and analysed by LC‐MS/MS. TREM2 in the conditioned media of primary human macrophages (black), primary murine microglia (white) and HEK293 cells stably expressing hTREM2 (grey) was digested with trypsin and the resulting peptides identified by mass spectrometry. The most frequent C‐terminal residue not consistent with trypsin digestion was H157 (from two donors/biological replicates of HEK293 cells; each replicate assayed in separate mass spectrometry laboratories; n = total number of peptides identified; where peptide sequences differ between species they are shown as human/mouse). Trypsin digestion (red sites) of band 1 provided almost complete coverage of macrophage TREM2 (bold), lacking only the peptide expected to have R52 at its C‐terminus. Non‐trypsin cleavage (blue) was observed predominantly at H157. The absence of the peptide with K42 at its C‐terminus suggests that N‐terminal truncation is responsible for generating band 2. Underlined: predicted secretion signal peptide. Peptides from the supernatants of HEK293 cells transiently expressing wild‐type (black) and H157Y (white) human TREM2, and co‐expressing human DAP12, were identified by mass spectrometry. The most common C‐terminus was residue 157 for both TREM2 isoforms (one biological replicate; n = total number of peptides identified; where WT and variant sequences vary they are shown as WT/variant). Schematic of the TREM2 protein. SP, signal peptide; IG domain, immunoglobulin domain; TM, transmembrane domain; triangles, N ‐glycosylation sites; arrow, site of proteolytic shedding; all numbers relate to amino acid positions. Source data are available online for this figure.

    Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).

    Techniques: Mass Spectrometry, Immunoprecipitation, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing, Variant Assay