Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 1. Chronic TREM2 activation increases DAM around plaques. (A) Schematic of experimental design. 6-mo-old 5XFAD mice were injected with AD- tau in the HC (bregma: −2.5 mm; lateral: −2.0 mm; depth: −2.2 mm) and overlying cortex (bregma: −2.5 mm; lateral: −2.0 mm; depth: −1.0 mm) and sacrificed 3 mo later to evaluate peri-plaque pathologies. 1 wk before AD-tau injection and every week following until sacrifice, 5XFAD mice were given i.p. injections of 80 mg/ml of the AL002a mouse TREM2 antibody (n = 13 female, n = 14 male) or the IgG control antibody (n = 14 female, n = 12 male). (B) Quantification of TREM2 antibody levels in terminal plasma for 5XFAD mice either chronically treated with the IgG control antibody or the TREM2 antibody. (C) Quantification of TREM2 antibody levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (D) Quantification of TREM2 antibody levels in cortical tissue for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. For acute treatment, there were n = 8 IgG control and n = 9 Trem2 antibody–treated age- and sex-matched mice, unless otherwise specified on graphs. (E) Representative images of ipsilateral hemisphere stained with CLEC7A+ microglia, P2RY12+ microglia, and Aβ from IgG control and TREM2 antibody–treated groups. Scale bars, 15 µm. (F) Quantification of mouse sTREM2 levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (G) Quantification

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Injection, Control, Clinical Proteomics, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 2. Chronic TREM2 activation with a TREM2 antibody results in no changes in Aβ plaque burden. (A) Representative images of Aβ plaques in 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Scale bar, 100 µm. (B–E) Quantification of Aβ staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of Aβ staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Scale bar, 100 µm. Significance was determined using a Student’s t test. ns, P > 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Control, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 3. Chronic TREM2 activation with a TREM2 antibody increases NP-tau pathology. (A) Representative images of ipsi- and contralateral hemi- spheres stained with AT8+ NP-tau pathology in AD-tau injected 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Repre- sentative images are from female mice. Scale bars, 100 µm. (B–E) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a Student’s t test. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Injection, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 4. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque NP-tau pathology and plaque-associated neuritic dystrophy, and acute TREM2 activation results in no changes in AD-tau uptake and degradation. (A) Representative images of BACE1+ and X34+ staining in ipsilateral HC. Scale bars, 20 µm. (B) Representative images of AT8+ and X34+ staining in ipsilateral HC. (C–F) Quantification of the number of BACE1+ staining within 15 µm of plaques in the ipsi- and contra- cortices (C and E) and hippocampi (D and F). (G–J) Quantification of the number of AT8+ staining within 15 µm of plaques in the ipsi- and contra- cortices (G and I) and hippocampi (H and J). (K) AD-tau uptake assay in TREM2 WT BMDMs. Results represent two independent ex- periments. (L) AD-tau degradation assay in TREM2 WT BMDMs. Results represent two independent experiments. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Degradation Assay, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 5. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque loss of synaptic marker synapsin. (A) Representative images of Synapsin+ and X34+ staining in ipsilateral HC. Scale bar, 7 µm. (B) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-HC. (C–E) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-cortex, contra-cortex, and HC. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a linear regression with sex as a covariate. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Marker, Staining, Control

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 <0.01, n =3, Student’s t -test.

Article Snippet: The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Construct, Transfection, Stable Transfection, Expressing, Immunoprecipitation, Control, Western Blot, Mutagenesis

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 <0.001, n =3 independent experiments, one-way ANOVA with Dunnett’s post hoc analysis. ( d ) Cell lysates were immunoprecipitated with the PS1 antibody Ab14 or normal IgG. TREM2, NCT and PS1-CTF were detected by immunoblotting. The levels of precipitated TREM2 WT and mutants were normalized to the input. * P <0.05, n =3, Student’s t -test.

Article Snippet: The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transfection, Stable Transfection, Expressing, Immunostaining, Marker, Immunoprecipitation, Western Blot

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: The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques:

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 <0.05, n =3, one-way ANOVA with Sidak post hoc analysis. ( b ) The level of endogenous TREM2 at the surface of microglial BV2 cells stably expressing PS1 (BV2-PS1) was determined by biotinylation. ** P <0.01, n =3, Student’s t -test. ( c ) BV2 cells were transfected with a scrambled control siRNA or PS1-targeting siRNAs for 72 h. The level of cell surface TREM2 was determined by surface biotinylation. * P <0.05, n =3, Student’s t -test. ( d ) Cell surface expression of TREM2 in HEK293-TREM2 cells with NCT overexpression, as determined by biotinylation. n =3, Student’s t -test.

Article Snippet: The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Cell Surface Biotinylation Assay, Stable Transfection, Expressing, Transfection, Control

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 <0.05, n =3, one-way ANOVA with Sidak post hoc analysis. ( c ) Phagocytosis of FAM-Aβ42 in BV2 cells following PS1 knockdown as determined by flow cytometry. n =3, Student’s t -test.

Article Snippet: The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

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

Journal: Brain, behavior, and immunity

Article Title: HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage

doi: 10.1016/j.bbi.2018.07.021

Figure Lengend Snippet: HX600 reduces Iba-1, phospho-p38 and TREM2 immunoreactivities in the ischemic brain. Quantitative analysis of the Iba-1, phospho-p38 and TREM2 immunoreactivities (A–C), and typical representative images of group receiving vehicle (D–F) or HX600 treatment (G–I) at time point 1 day after the insult. Immunoreactivities of Iba-1 and phospho-p38 were analyzed from the peri-ischemic area, and TREM2 at the lesion site. Scale bar 100 μm. Data presented as mean ± SD. Unpaired two-tailed t-test, n = 8 in each group. *p < 0.05 ***p < 0.001.

Article Snippet: For phospho-p38 and TREM2 stainings biotinylated secondary antibody (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) was used, followed by reaction with avidin-biotin complex reagent (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) according to the instructions provided by the manufacturer.

Techniques: Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: TREM2-CLuc-IRES-TYROBP-NLuc split-Renilla luciferase reporter system. The TREM2-CLuc-IRES-TYROBP-NLuc vector contains CMV immediate early (IE) promoter, the C terminus of the Renilla luciferase gene fused to the cytoplasmic region of TREM2 (TREM2-CLuc), IRES, and the N-terminal region of the Renilla luciferase gene fused to the N-terminal region of TYROBP (TYROBP-NLuc) (A). The unique restriction sites for cloning are depicted. Ampr, ampicillin resistance gene; Neor, neomycin resistance gene. In a resting state, wild-type TREM2 and TYROBP show limited coupling. Upon stimulation with agonistic ligand (such as dimerizing anti-TREM2 antibody), TREM2 interacts with TYROBP, which reconstitutes luciferase activity by coupling of the C- and N-terminal regions of the split-Renilla luciferase gene (B).

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Luciferase, Plasmid Preparation, Clone Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: Anti-TREM2 antibody, but not Aβ or anti-TYROBP antibody (Ab), induces dose-dependent increase in the TREM2-TYROBP interaction in HEK293 cells. HEK293 cells were transfected with internal control reporter pRL-TK, TREM2-CLuc-TYROBP negative control, or our TREM2-CLuc-IRES-TYROBP-NLuc plasmid prior to stimulation with ligand. Time-course data (A, C, E, and G) were assessed for luminescence over time by AUC analysis (B, D, F, and H) in RLU for n = 3 for each group. Error bars represent S.D. ααα denotes p < 0.001 versus all other groups. * denotes p < 0.05 versus TREM2/PBS as determined by one-way ANOVA and Tukey's post-test.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Transfection, Negative Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: Quantification of TREM2·TYROBP complex by “sandwich” ELISA. HEK293 cells were transfected with TREM2-CLuc-IRES-TYROBP-NLuc followed by anti-TREM2 antibody (Ab) stimulation to induce TREM2 coupling to TYROBP and protein cross-linking with DTBP and phosphatase inhibitor sodium pervanadate (Na3O4V) (A). Following cell lysis, protein was incubated in anti-TYROBP-coated wells and labeled for TREM2 with biotinylated anti-TREM2·HRP-conjugated streptavidin complex for quantification of TREM2·TYROBP protein complexes. Average (Ave) OD was quantified by ELISA. Data represent the arithmetic difference between a given treatment condition and the average OD of untransfected controls (n = 4 per group) (B). Significant differences were determined by one-way ANOVA followed by Tukey's post-test. Error bars represent S.D. †† and ††† denote p < 0.01 and p < 0.001, respectively, versus untransfected cells. ### denotes p < 0.001 versus unstimulated cells transfected with TREM2-TYROBP. ** and *** denote p < 0.01 and 0.001, respectively, versus those indicated.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Sandwich ELISA, Transfection, Lysis, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: Anti-TREM2 antibody stimulation enhances phagocytic capability of BV-2 microglial cell line. Cells were gated based on unstained cells (A), anti-TREM2 antibody-treated cells (B), or S. aureus-treated cells (C). Cells that were treated with anti-TREM2 antibody (Ab) and S. aureus particles are shown in D. The phagocytic cell population (percentage is indicated above plot) was further separated into phagocytic Lo versus Hi populations (E–G) (52). In H, relative distributions of the phagocytic cell populations into low versus high indices are represented by FlowJo software and graphical analysis. Significant differences in low or high populations were determined by one-way ANOVA followed by Tukey's post-test. Error bars represent S.D. #, ##, and ### denote p < 0.05, 0.01, and 0.001, respectively, versus those groups indicated.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Software

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody (Ab) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an ADP-Glo kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p < 0.001 versus untransfected cells, and ### denotes p < 0.001 versus TREM2 WT group with antibody stimulation + SYK inhibitor.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Activation Assay, Transfection, Expressing, Immunoprecipitation, Activity Assay, Kinase Assay

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: The T66M mutation in TREM2 elicits enhanced TREM2 coupling to TYROBP in HEK293 cells. HEK293 cells were transfected with TREM2-CLuc-IRES-TYROBP-NLuc plasmid prior to stimulation with ligand. Time-course data (A, C, and E) were assessed for AUC (B, D, and F) in RLU for n = 3 for each group. Significant differences were determined by one-way ANOVA followed by Tukey's post-test. Error bars represent S.D. † and ††† denote p < 0.05 and 0.001, respectively, versus unstimulated WT TREM2-transfected cells. ** and *** denote p < 0.01 and 0.001, respectively. Ab, antibody.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Mutagenesis, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: Reduced cell surface expression of TREM2 T66M mutant. A and B, flow cytometry analysis of TREM2 signal (x axis) in untransfected (blue) and transfected cells (red) with TREM2 WT, R47H, S116C, or T66M after gating with forward and side scatter profiles for live cells (not shown) (A). The numbers in A show the percent fraction of TREM2+ cells in transfected cells, and B shows profiles of TREM2+ cells after gating in A. C and D, the same experiment was performed in permeabilized cells for untransfected (blue) and transfected cells (red). The numbers in C show the percent fraction of TREM2+ cells in transfected cells, and D shows profiles of TREM2+ cells after gating in C. Average fluorescence intensity was measured for TREM2+ cells for each group in unpermeabilized (E) and permeabilized conditions (F). Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. * and ** denote p < 0.05 and 0.01 versus TREM2 WT group. ††† denotes p < 0.001 versus untransfected cells (E and F).

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Expressing, Mutagenesis, Flow Cytometry, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

doi: 10.1074/jbc.M116.759159

Figure Lengend Snippet: Primer sequences for PCR amplification For, forward; Rev, reverse.

Article Snippet: To label TYROBP protein cross-linked with TREM2, TYROBP-bound protein was labeled with a biotinylated anti-human TREM2 antibody (goat polyclonal IgG, BAF1828, R&D Systems)·HRP-conjugated streptavidin (21126, Life Technologies) complex (0.5 μg/ml each) in 0.1% Triton X-100 in PBS for 1 h at room temperature.

Techniques: Amplification, Sequencing