Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: ELS reduces expression of TREM2 and CD11b while increasing CX3CR1 in P17 microglia. (A) Scatter plots for quantifying Trem2 positive microglia in CTL, LB and UPS. (B) Percentage of TREM2-positive microglia. Median fluorescence intensity (MFI) plots and surface expression of CD11b (C-D), CD45 (E-F) and CX3CR1 (G-H). CTL-males: n = 16, CTL-females: n = 14, LB males: n = 10, LB-females: n = 9, UPS-males: n = 12, UPS-females: n = 16. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Expressing, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: Trem2 is essential for normal phagocytic activity. (A-B) Effects of Trem2 genotype (WT, Hets, Ko) on ex vivo phagocytic activity in microglia isolated from the hippocampus of 17-day old pups (n = 10–17 pups per group, 50 % females). (C) Representative confocal images (top row) and Imaris models (middle and lower rows) of microglia from Trem2 wildtype (WT), heterozygous (Hets) and knockout P17 littermates. Staining for Iba1 (green), CD68 (blue), and PSD95 (red). Middle row: reconstruction of Iba1 & CD68 staining. Lower row: reconstruction of CD68 and PSD95 staining inside microglia. Effects of Trem2 genotype on microglial volume (D), CD68 volume inside microglia (E), number of PSD95 puncta in microglia (F) and PSD95 puncta inside CD68 (G), n = 5 cells per mouse and 4–5 mice per group, 50 % females). Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Activity Assay, Ex Vivo, Isolation, Knock-Out, Staining

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: ( A–D ) mRNA levels of indicated genes in the SVFs of eWAT from ND, HFD-veh and HFD-STF mice, assessed by qRT-PCR. The results are expressed as fold change and are representative of three independent experiments. ( E–G ) Immunofluorescent staining of CD9 and Trem2 in the eWAT sections of DIO mice treated with vehicle or STF. Representative images shown ( E ) and percentages of the numbers of CD9-positive cells ( F ) and Trem2-positive cells ( G ) over total cells/field. ( H ) Analysis of CD9-positive ATMs (F4/80 + CD11b + CD9 + ) from eWAT SVFs of mice with an overlay of gated CD9 marker for ND (blue), HFD-Veh (yellow) and HFD-STF (pink) shown. Data in Y-axis was presented as ‘Normalized to mode’ for percentages. Complete gating path was shown in . ( I ) Analysis of Trem2-positive ATMs (F4/80 + CD11b + Trem2 + ) from eWAT SVFs of mice with an overlay of gated Trem2 marker for ND (blue), HFD-Veh (yellow) and HFD-STF (pink) shown. Data in Y-axis was presented as ‘Normalized to mode’ for percentages. Complete gating path was shown in . ( J ) Flow cytometry analysis of CD9 + and Trem2 + ATMs from eWAT SVFs of ND, HFD-Veh and HFD-STF mice, gated against CD9 and Trem2 antibodies. Complete gating path was shown in . ( K–L ) mRNA levels of indicated proinflammatory genes in the indicated cells sorted from SVFs of eWAT of DIO mice, assessed by qRT-PCR: for CD9 + vs CD9 - ATMs ( K ) and for Trem2 + vs Trem2 - ATMs ( L ). The results are expressed as fold change and are representative of three independent experiments. M-N. mRNA levels of indicated proinflammatory cytokine/chemokine genes in the CD9 + ATMs (F4/80 + CD11b + CD9 + ) ( M ) and the Trem2 + ATMs (F4/80 + CD11b + Trem2 + ) ( N ) sorted from SVFs of eWAT of DIO mice treated with vehicle or STF, assessed by qRT-PCR. The results were expressed as fold change and are representative of three independent experiments. O-P. Flow cytometry analysis of ATMs from SVFs of the eWAT from 20-week HFD-fed mice. SVFs were cultured and treated with 0.01% DMSO control or STF (30 μM) for 20 hr followed by staining and flow analysis, gated against F4/80 and CD11b antibodies ( O ) and further gated against Trem2 antibody ( P ). Data in Y-axis in P was presented as ‘Normalized to mode’ for percentages. The gating path followed the one as . Q-S. mRNA levels of indicated genes in CD9 + ATMs ( Q and R ) and in CD11C + ATMs ( S ) sorted from SVFs cultured and treated with DMSO or STF as in O-P, assessed by qRT-PCR. Data were expressed as mean ± SEM and analyzed using the unpaired two-tailed Student’s t-test between two samples or ANOVA with multiple comparisons. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Quantitative RT-PCR, Staining, Marker, Flow Cytometry, Cell Culture, Control, Two Tailed Test

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: ( A–C ) mRNA levels of indicated genes in the SVFs of iWAT from ND, HFD-veh and HFD-STF mice, assessed by qRT-PCR. The results are expressed as fold change and are representative of three independent experiments. ( D-D’ ) Representative images ( D ) of immunofluorescent staining of CD9 (red) in the eWAT sections of ND- and HFD-fed mice and quantification ( D’ ). DAPI (blue) for nuclear staining. ( E-E’ ) Representative images ( E ) of immunofluorescent staining of Trem2 (red) in the eWAT sections of ND- and HFD-fed mice and quantification ( E’ ). DAPI (blue) for nuclear staining. ( F ) Representative images of immunofluorescent staining of CD9 (red) and Trem2 (green) in the BAT sections of HFD-fed mice treated with Veh or STF. DAPI (blue) for nuclear staining. ( G–H ) Representative images of immunofluorescent staining in the iWAT sections of HFD-fed mice treated with Veh or STF ( G ) and quantification ( H ). CD9 (red), Trem2 (green), and DAPI for nuclear staining (blue). Data are the mean ± SEM. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Quantitative RT-PCR, Staining

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: Characterization of and the effect of STF treatment on CD9 + and Trem2 + ATMs in DIO mice. The percentage, total cell number, and density of subpopulations of CD9 + ATMs (A), Trem2 + ATMs (B), and CD9 + Trem2 + ATMs(C) in eWAT from mice with ND, HFD-Veh, or HFD-STF. Data in a-c were obtained from two batches of four mice each and were expressed as mean ± SEM and analyzed using the unpaired two-tailed Student’s t-test between two samples or ANOVA with multiple comparisons. ∗p <0.05, ∗∗p <0.01, and ∗∗∗p <0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Significance Assay

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: ( A ) Flow cytometry analysis of CD63 signal in CD9 + ATMs from eWAT SVFs of DIO mice, gated against CD9 and CD63 antibodies sequentially following the gating strategy shown in . Data in Y-axis was presented as ‘Normalized to mode’” for percentages. ( B ) Flow cytometry analysis of CD9 + and CD63 + ATMs from eWAT SVFs of ND, HFD-Veh and HFD-STF mice, gated against CD9 and Trem2 antibodies simultaneously following the gating strategy shown in . ( C ) Flow cytometry analyses of Trem2 signal in CD9 + ATMs (left panel) and of CD9 signal in Trem2 + ATMs (right panel) from eWAT SVFs of DIO mice, gated against CD9 and Trem2 antibodies sequentially (left panel) or against Trem2 and CD9 sequentially (right panel) following the gating strategy shown in . Data in Y-axis was presented as ‘Normalized to mode’ for percentages. Data are the mean ± SEM. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Flow Cytometry

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: ( A ) Overlay of the flow cytometric analysis of percentages of CD9-positive signals among CD11C- hi (pink), CD11C + (yellow), and CD11C - (blue) ATMs from eWAT SVFs of DIO mice. Data in Y-axis was presented as ‘Normalized to mode’ for percentages. ( B–C ) Overlay of the flow cytometric analysis of percentages of CD9-positive ( B ) and CD9-and Trem2-doubly positive ( C ) signals among CD11C + (yellow) and CD11C - (blue/green) ATMs from eWAT SVFs of DIO mice. ( D–F ) Overlay of the flow cytometric analysis of percentages of CD11-positive signals among CD9 hi, +, and – ATMs ( D ), Trem2 + and – ATMs ( E ), and CD9/Trem2 + and – ATMs ( F ) from eWAT SVFs of DIO mice. Data in Y-axis was presented as ‘Normalized to mode’ for percentages. The gating strategies for A-F were shown in . ( G ) Flow cytometry of CD9 + and CD11C + ATMs from eWAT SVFs of DIO mice treated with vehicle or STF, gated against CD9 and CD11C antibodies. Complete gating path was shown in . ( H ) mRNA levels of indicated proinflammatory cytokine/chemokine genes in the indicated ATM subpopulations sorted from SVFs of eWAT of DIO, assessed by qRT-PCR. The results were expressed as fold change and were representative of three independent experiments. Data were expressed as mean ± SEM and analyzed using the unpaired two-tailed Student’s t-test between two samples or ANOVA with multiple comparisons. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in male obese mice

doi: 10.7554/eLife.100581

Figure Lengend Snippet: Flow cytometry analysis of relationship between ‘M1-like’ ATMs and CD9 + /Trem2 + ATMs. (A) The percentages of cells expressing CD9, Trem2 or both CD9 and Trem2 markers in CD11c + or CD11c - ATMs from eWATs of DIO mice. (B) The percentages of cells expressing CD11C marker in CD9 + , Trem2 + , or CD9 + Trem2 + ATMs versus their negative counterparts from eWATs of DIO mice. (C) The percentage, total cell number, and density of CD9 + CD11c + ATMs from eWATs of DIO mice treated with vehicle or STF. Data in a-c were obtained from two batches of four mice each and were expressed as mean ± SEM and analyzed using the unpaired two-tailed Student’s t-test between two samples or ANOVA with multiple comparisons. ∗p <0.05, ∗∗p <0.01, and ∗∗∗p <0.001.

Article Snippet: For Immunofluorescence staining, the eWAT and iWAT paraffin sections (the section thickness is 5–10 μm) were blocked with 1% BSA, 0.2% Triton PBS, then incubated with primary antibody F4/80 (Cat#:70076, RRID: AB_2799771 , Cell Signaling Technology), CD9 (Cat#:ab223052, RRID: AB_2922392 , Abcam), Trem2 (Cat#: MAB17291, RRID: AB_2208679 , R&D Systems) UCP1(Cat#:ab10983, RRID: AB_2241462 , Abcam) overnight at 4 °C after deparaffinization.

Techniques: Flow Cytometry, Expressing, Marker, Significance Assay

Journal: medRxiv

Article Title: MS4A4A modifies the risk of Alzheimer disease by regulating lipid metabolism and immune response in a unique microglia state

doi: 10.1101/2023.02.06.23285545

Figure Lengend Snippet: A. MS4A4A and TREM2 reside on the plasma membrane in microglia. Prior work demonstrates that rs1582763 is associated with elevated CSF sTREM2 and reduced AD risk (termed: protective) and rs6591561 is associated with reduced CSF sTREM2 and increased AD risk (termed: risk) B. Trans-eQTL analyses for rs1582763 and rs6591561 were performed using data derived from tissue samples representing three cortical areas in three independent cohorts (ROSMAP, frontal; Knight ADRC, parietal; Mayo, temporal). C. Trans-eQTL analyses were performed in human microglia clusters from single nuclei RNA-seq. D. Functional genomics were applied to define the impact of rs1582763 and rs6591561 on microglia function using iTF-microglia. E. Summary of major findings.

Article Snippet: For detection, wells were incubated with a Trem2 biotinylated detection antibody (R&D Systems, BAF1729, [0.25μg/ml]) for 1 hour at RT on an orbital shaker.

Techniques: Clinical Proteomics, Membrane, Derivative Assay, RNA Sequencing, Functional Assay

Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 1. Microglial TREM2 is required for the reduction in poly-GA proteins (A) GO analysis of the biological processes and molecular functions of upregulated differentially expressed genes in poly-GA-expressing cortices. Three bio- logical repeats were performed.

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Expressing

Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 3. Activation of the NLRP3 inflammasome inhibits poly-GA clearance by promoting TREM2 cleavage (A) Fluorescence images of poly-GA proteins in NLRP3 and TREM2 double- or single-knockout motor cortices. (B) Quantification of the area of GFP-GA signal in (A). n = 3, 3, 3, and 5 mice in each group; at least 3 cortex sections per mouse for statistics. (C) Immunoblot of mature TREM2 levels. BV2 cells were treated with IL-1b (10 ng/mL) for 12 h. (D) Immunoblot of cleaved TREM2 (sTREM2) levels in the cell medium of BV2 cells. (E) Immunoblotting showed that GI 254023X inhibited sTREM2 production. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/ mL, 12 h). (F) ELISA of sTREM2 levels in cerebrospinal fluid of control and poly-GA mice. n = 3 mice per group. (G) Immunoblot of p-DAP12 levels. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/mL, 12 h). Cell lysates were immu- noprecipitated by the anti-DAP12 antibody and immunoblotted with the antibody 4G10. (H and I) Quantification of immunofluorescent area of poly-GA. Wild-type or TREM2/ primary microglia were pretreated with GI 254023X (10 mM) and IL-1b (10 ng/mL) and incubated with FLAG-GA-His aggregates for 12 h totally. n = 3 independent experiments. (J) Immunoblot of active ADAM10. ATP1A1 indicated the loading control of BV2 cell membrane. Unless otherwise specified, at least three independent experiments were performed. t test in (F), (H), and (I). One-way ANOVA in (B).

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Activation Assay, Fluorescence, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Membrane

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: Figure 1. TREM2 expression, glycosylation and proteolysis.

Article Snippet: Murine TREM2 was immunoprecipitated with a rat anti-mTREM2 monoclonal (MAB1729, R&D Systems).

Techniques: Expressing, Glycoproteomics

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: Figure 2. Shedding of glycosylated TREM2 NTF is sensitive to inhibitors of ADAM10 and matrix metalloproteinases.

Article Snippet: Murine TREM2 was immunoprecipitated with a rat anti-mTREM2 monoclonal (MAB1729, R&D Systems).

Techniques:

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: Figure 6. Knock-down of ADAM10 is less effective at reducing the shedding of H157Y TREM2.

Article Snippet: Murine TREM2 was immunoprecipitated with a rat anti-mTREM2 monoclonal (MAB1729, R&D Systems).

Techniques: Knockdown