Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) Schematic of the phage display workflow. The extracellular domain of TREM2 was used as the target for selection from a cysteine-constrained cyclic peptide M13 phage library. Four rounds of biopanning, including binding, washing, elution, and amplification, were performed to enrich TREM2-binding clones. (B) Top enriched peptide sequences identified from rounds 2-4. Filled circles indicate detection of a given sequence in the corresponding round, while open circles indicate absence. All sequences conform to a cysteine-constrained cyclic peptide scaffold. (C) Phage ELISA validation of selected peptides. Binding signals are presented as the ratio of signal obtained in TREM2-coated wells relative to control wells lacking protein (E/C). Values above 1 indicate preferential binding to TREM2. Data are shown as mean ± SEM (n=3).

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Selection, Binding Assay, Amplification, Clone Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Control

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) IL-1β secretion in human iPSC-derived microglia following Aβ 1-42 oligomer challenge in the presence of TREM2-6 or TREM2-12 (5, 10, and 25 µM). VG-3927 (5 μM) was used as a positive control. Data are normalized to the Aβ-treated vehicle control and expressed as percent change. (B) Loss of peptide-mediated suppression of IL-1β secretion in TREM2 knockout (KO) microglia confirms TREM2-dependent activity. (C) Modulation of secreted ApoE levels in human microglia following Aβ exposure and peptide treatment (25 µM). ApoE levels are normalized to vehicle-treated controls. Data are presented as mean ± SD (n = 5). Statistical significance was determined by one-way or two-way ANOVA with Dunnett’s post hoc test. ns , not significant; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) relative to vehicle treatment.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Derivative Assay, Positive Control, Control, Knock-Out, Activity Assay

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: Quantification of PSD95 levels in human iPSC-derived neuron-microglia co-cultures following Aβ 1-42 oligomer exposure and treatment with TREM2-6 or TREM2-12 (5, 10, and 25 µM). VG-3927 (5 μM) was used as a positive control. PSD95 levels are expressed as percent rescue relative to Aβ-treated vehicle controls. Data are presented as mean ± SD (n = 5). Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) relative to vehicle treatment.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) Time-dependent root-mean-square deviation (RMSD) of the TREM2 receptor in the free form (cyan line) and in complex with T6 (purple line) and T12 (yellow line) over a 100 ns MD simulation. (B) RMSD of the T6 and T12 over a 100-ns MD simulation.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques:

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: B) Structural snapshot clustered from the late stage of the MD simulation of T12 (A) and T6 (B) complexed with TREM2. The receptor is shown in grey cartoon, T12 is shown in blue sticks, and T6 is shown in orange sticks. (C and D) Views of the binding interface between T12 (C) / T6 (D) and TREM2 with the key interacting residues on both sides and yellow dashed lines that demonstrate stable hydrogen bonds and salt bridges.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine- 13 C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D , E , F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Labeling

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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 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 < 1 h, and that little was found in cytosol or nuclear cellular fractions (green triangles & purple inverted triangles). Data plotted as mean ± SEM; n = 4 replicates. Source data are available online for this figure.

Article Snippet: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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‐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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: 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 < 0.05; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

Techniques: Concentration Assay, Expressing, Variant Assay

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: A–E (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. F, G 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

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

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: A, B 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. C 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

Techniques: Western Blot, Quantitation Assay, Inhibition

Journal: EMBO Molecular Medicine

Article Title: TREM 2 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: A, B 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: Samples and a 7‐point standard curve with recombinant human TREM2 (Sino Biological) diluted in cell culture medium were generated and added to plates for 2 h at room temperature.

Techniques: Stable Transfection, Expressing