human trem2  (Sino Biological)


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    TREM 2 Matched ELISA Antibody Pair Set Human
    Description:
    Each vial contains 100 ng of recombinant Human TREM 2 TREM2 Reconstitute with 1 mL detection antibody dilution buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve using 2 fold serial dilutions in sample dilution buffer and a high standard of 3500 pg mL is recommended
    Catalog Number:
    SEK11084
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    Elisa pair set
    Reactivity:
    Human
    Product Aliases:
    TREM-2 Matched ELISA Antibody Pair Set Human, Trem2a Matched ELISA Antibody Pair Set Human, Trem2b Matched ELISA Antibody Pair Set Human, Trem2c Matched ELISA Antibody Pair Set Human
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    Sino Biological human trem2
    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) <t>TREM2</t> 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.
    Each vial contains 100 ng of recombinant Human TREM 2 TREM2 Reconstitute with 1 mL detection antibody dilution buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve using 2 fold serial dilutions in sample dilution buffer and a high standard of 3500 pg mL is recommended
    https://www.bioz.com/result/human trem2/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human trem2 - by Bioz Stars, 2021-04
    93/100 stars

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    1) Product Images from "TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant"

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

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201707673

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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%.
    Figure Legend 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%.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    Related Articles

    Cell Culture:

    Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant
    Article Snippet: .. TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2. .. Rabbit anti‐TREM2 monoclonal capture antibody from Sino Biological's ELISA was coated at 2 μg/ml on MSD L15XA plates.

    Sandwich ELISA:

    Article Title: Glial activation and inflammation along the Alzheimer’s disease continuum
    Article Snippet: They were determined using Innotest β-Amyloid (1-42), Innotest T-tau Ag, and Innotest P-tau (181P)(Fujirebio, Ghent, Belgium), respectively. .. CSF sTREM2 was also analyzed using a sandwich ELISA as described earlier [ ] with some modifications; the plates were coated over night with the capture antibody (0.25 μg/ml; AF1828, R & D Systems, MN, USA) and samples incubated for 2 h prior to TREM2 detection with a rabbit-monoclonal anti-human TREM2 antibody (0.5 μg/ml; SEK11084, Sino Biologics, Beijing, China). .. The QuickPlex SQ 120 system from Meso Scale Discovery (MSD, MD, USA) was used to measure YKL-40, MCP-1, and fractalkine in a U-plex format and clusterin in an R-plex format, where YKL-40 and clusterin were in a singleplex setup and MCP-1 and fractalkine were in the same multiplex setup.

    Incubation:

    Article Title: Glial activation and inflammation along the Alzheimer’s disease continuum
    Article Snippet: They were determined using Innotest β-Amyloid (1-42), Innotest T-tau Ag, and Innotest P-tau (181P)(Fujirebio, Ghent, Belgium), respectively. .. CSF sTREM2 was also analyzed using a sandwich ELISA as described earlier [ ] with some modifications; the plates were coated over night with the capture antibody (0.25 μg/ml; AF1828, R & D Systems, MN, USA) and samples incubated for 2 h prior to TREM2 detection with a rabbit-monoclonal anti-human TREM2 antibody (0.5 μg/ml; SEK11084, Sino Biologics, Beijing, China). .. The QuickPlex SQ 120 system from Meso Scale Discovery (MSD, MD, USA) was used to measure YKL-40, MCP-1, and fractalkine in a U-plex format and clusterin in an R-plex format, where YKL-40 and clusterin were in a singleplex setup and MCP-1 and fractalkine were in the same multiplex setup.

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    Sino Biological human trem2
    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) <t>TREM2</t> 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.
    Human Trem2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human trem2/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human trem2 - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

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    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

    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: TREM2 meso scale discovery immunoassay A sandwich immunoassay quantified the concentrations of shed TREM2 in cell culture supernatants from macrophages and HEK293 cells over‐expresssing human TREM2.

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