sheep trem2  (R&D Systems)

Journal: Cardiovascular Research

Article Title: Single-cell to pre-clinical evaluation of Trem2 , Folr2 , and Slc7a7 as macrophage-associated biomarkers for atherosclerosis

doi: 10.1093/cvr/cvaf210

Figure Lengend Snippet: Comparative analysis of gene expression and sTREM2 levels in mouse and human samples. ( A ) Venn diagram illustrating the overlap of gene expression profiling results from mouse aorta samples analysed by TRAP-Seq and scRNA-Seq, and human coronary arteries analysed by scRNA-Seq, revealing a subset of genes enriched in macrophages. The analysis identifies 388 macrophage-enriched genes, of which 164 are membrane proteins, 33 are metabolic proteins, and 54 are receptor proteins. Sample sizes: TRAP-Seq n = 6 mice, mouse scRNA-Seq n = 3 mice, and human scRNA-Seq n = 4 patients. ( B ) Detailed lists of identified proteins categorized into metabolic proteins and receptor proteins from A . ( C ) Expression of Trem2 , Slc7a7 and Folr2 in TRAP-Seq and scRNA-Seq of mouse aorta. For TRAP-Seq, values represent the average transcripts per million across IP replicates, while for scRNA-Seq, they correspond to cluster pseudobulk counts per million. Gene expression for each method is scaled per gene from 0 to 1, with 1 representing the maximum observed expression. Sample sizes are as indicated in panel A . ( D ) Quantification of soluble (s)TREM2 in the plasma of mice carrying (+) or not carrying (−) the hypercholesterolemic LDLR −/− ApoB 100/100 mutations. The mice were subjected to either a regular diet (0) or a high-fat diet (HFD) for 1 or 3 months. Data are presented for both 12-month-old and 21-month-old mice, illustrating the impact of diet and age. The Wilcoxon rank sum test was used for comparing groups. Sample size (mice per group) at 12 months was 7 (−; HFD 0), 5 (+; HFD 0), 5 (+; HFD 1), 7 (−; HFD 3) and 4 (+; HFD 3), and at 21 months was 25 (−) and 30 (+). ( E , F ) Normalized protein expression (NPX levels of TREM2 in ( E ) human atherosclerotic tissue and ( F ) plasma from individuals of the BiKE cohort in individuals with asymptomatic (AS) and symptomatic (S) carotid stenosis. NPX was calculated as recommended by Olink and is on a log 2 scale in arbitrary units (one NPX difference corresponds to 2-fold in protein concentration). Each dot represents an individual patient's sTREM2 levels, with 50 patients in each group. Statistical significance was measured using a two-sided Student’s t -test with unequal variance. ( G ) Heatmap of Pearson correlation coefficients between plasma and tissue TREM2 levels (atherosclerotic aortic arterial wall = aorta, liver, and mammary artery = m.artery) in 165 coronary artery disease patients from the STARNET cohort. ( H ) Pearson correlation matrix showing relationships between plasma sTREM2, C-reactive protein, serum cholesterol, low-density lipoprotein cholesterol, and triglycerides in 20 individuals from the BiKE cohort. For both panels, the colour scale represents the Pearson correlation coefficient ( r ), ranging from −1 to +1. Asterisks denote statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The plates were washed five times with washing buffer, followed by a 1-h incubation at RT with a biotinylated mouse anti-TREM2 antibody (BAF1729, 1:3000, R&D Systems).

Techniques: Gene Expression, Membrane, Expressing, Clinical Proteomics, Protein Concentration

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: Elevated TREM2 protein and mRNA levels in activated microglia in the hippocampus of 5 × FAD mice during disease progression. (A) Analysis of the mRNA expression of TREM2 in the hippocampi of 1 month-old to 18–20 months-old 5 × FAD mice and aged-matched WT mice. The expression of TREM2 mRNA is presented as the mean ± S.E.M. and is shown as the relative expression level according to the 2 Δ Δ Ct method. The data were analyzed via one-way analysis of variance followed by Bonferroni post hoc correction. # p < 0.05, ## p < 0.01, ### p < 0.001, 5 × FAD young group (3–4 months old) vs. other 5 × FAD groups. * p < 0.05, ** p < 0.01, *** p × 0.001, WT vs. 5 × FAD. (B) Representative western blot of TREM2 in whole hippocampal lysate, expressed as the mean ± S.E.M. (C) Representative confocal photomicrographs of the whole hippocampus. Note the increased immunoreactivity of TREM2 (red) in the hippocampus, especially in the CA1 region, of 5 × FAD mice. The expression increased with age. Scale bar = 200 μm. (D) Dual-label immunofluorescence image showing TREM2 (red) with microglia (green). Nuclei are stained with DAPI (blue). Notably, the increased immunoreactivity of TREM2 (red) was mostly associated with activated microglia (Iba1, green) rather than with resting microglia. Scale bar = 10 μm. (E) Immunohistochemistry (IHC) showing Aβ deposits stained with 6E10 in WT mice and 5 × FAD mice. Scale bar = 100 μm. (F) Dual-label immunofluorescence showing that TREM2 (red) is expressed mainly in plaque-associated areas (6E10, green). Scale bar = 10 μm. ns, not significant; TREM2, triggering receptor expressed on myeloid cells-2; WT, wild type; Iba1, ionized calcium-binding adapter molecule 1.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Binding Assay

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: Upregulation of PU.1 protein and mRNA expression in the hippocampus of 5 × FAD mice during disease progression and triggering receptor expressed on myeloid cells-2 (TREM2) coexpression in microglia. (A) . Single-label immunohistochemistry (IHC) for the transcription factor PU.1 alone in the hippocampi of Alzheimer’s disease (AD) and control mice. Scale bar = 100 μm. (B) Representative western blot of PU.1 in whole hippocampal lysate, expressed as the mean ± S.E.M. (C) Analysis of the mRNA expression of PU.1 in the hippocampus from 1 to 18–20 months-old 5 × FAD mice and aged-matched wild type (WT) mice. The expression of PU.1 is presented as the mean ± S.E.M. and is indicated as the relative expression level via the 2 ΔΔCt method. The data were analyzed via one- or two-way analysis of variance followed by Bonferroni post hoc correction. # p < 0.05, ## p < 0.01, ### p < 0.001, 5 × FAD young group (3–4 months old) vs. the other 5 × FAD groups. * p < 0.05, ** p < 0.01, *** p < 0.001, WT vs. 5 × FAD.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Expressing, Biomarker Discovery, Immunohistochemistry, Control, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: The positive correlation between triggering receptor expressed on myeloid cells-2 (TREM2) gene expression and PU.1 in the microglia of 5 × FAD mice during chronic neurodegeneration. (A) A significant correlation was found between the mRNA and protein expression of TREM2 and that of PU.1 in 5 × FAD mice across the age groups. (B) Linear regression analysis revealed that total TREM2 protein levels in the hippocampus were positively correlated with PU.1 levels. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Analysis of coexpression of TREM2 (red) and PU.1 (green) in the hippocampi of 5 × FAD mice and aged-matched wild type (WT) mice by double immunofluorescence. Scale bar = 20 μm.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Gene Expression, Expressing, Immunofluorescence

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: Aβ induced the upregulation of triggering receptor expressed on myeloid cells-2 (TREM2) and PU.1 expression in microglia in vitro . Treatment with 1 μM oAβ (A) or fAβ (B) was conducted for various durations in cultured BV-2 cells. (C,D) Representative single-label immunofluorescence staining of TREM2 in primary microglial cells after treatment with different doses of Aβ for 24 h. The values are expressed as the mean fluorescence intensity of TREM2. The data are expressed as relative fold changes compared with the controls. The values are expressed as the means ± S.E.M.s. Scale bar =10 μm. The data were analyzed via one-way analysis of variance followed by Bonferroni post hoc correction. (E) Quantitative real-time PCR analysis of TREM2 and PU.1 mRNA in primary microglia after 1 μM oAβ treatment at various time points. The expression of TREM2 is presented as the mean ± S.E.M. and is presented as the relative expression level according to the 2 ΔΔCt method. The data were analyzed via one-way analysis of variance followed by Bonferroni post hoc correction. Aβ, amyloid beta. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Expressing, In Vitro, Cell Culture, Immunofluorescence, Staining, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: PU.1 promotes triggering receptor expressed on myeloid cells-2 (TREM2) gene expression at the mRNA and protein levels. (A–D) The effects of lentiviral particles on TREM2 and PU.1 gene expression in BV2 cells were confirmed by western blotting and qRT–PCR. The protein level is expressed as the relative level in the untreated cells, while the mRNA level is presented as the mean ± S.E.M. and is indicated as the relative expression level according to the 2 ΔΔCt method; * p < 0.05, ** p < 0.01, *** p < 0.001, ## p < 0.01. The columns represent the results of the quantitative analysis of the expression of TREM2 and PU. (E) Immunofluorescence staining further revealed that PU.1 shRNA reduced the expression of TREM2 (red) and that the PU.1-overexpressing virus (PU.1 OE virus) increased the expression of TREM2. Scale bar = 10 μm. (F) The downregulation of TREM2 by PU.1 shRNA was also confirmed in primary microglia.Scale bar = 10 μm. EGFP = vector control. The values are expressed as the means ± S.E.M.s and were analyzed via ANOVA with Bonferroni post hoc correction. shRNA, short hairpin RNA.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Gene Expression, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, shRNA, Virus, Plasmid Preparation, Control

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin immunoprecipitation (ChIP) assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Binding Assay, In Vitro, In Vivo, Activity Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Clone Assay, Plasmid Preparation, Construct, Transfection, Expressing, Luciferase, Reporter Assay

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: PU.1-dependent Aβ-mediated triggering receptor expressed on myeloid cells-2 (TREM2) expression upregulation in microglia. Microglial BV2 cells were transduced with a lentiviral vector encoding PU.1 short hairpin RNA (shRNA) or PU.1 sequences or control virus. After treatment with different viruses, the cells were treated with 1 μM oAβ (A) or fAβ (B) for 24 h. (A,B) Representative western blot of TREM2 protein levels upon oAβ or fAβ stimulation. Loss of PU.1 abrogated Aβ-induced TREM2 upregulation in microglia.oAβ, oligomeric amyloid beta; f Aβ, fiber amyloid beta. * p < 0.05, ** p < 0.01, *** p < 0.001, ## p < 0.01.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Expressing, Transduction, Plasmid Preparation, shRNA, Control, Virus, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: The impairment of microglial phagocytosis by triggering receptor expressed on myeloid cells-2 (TREM2) and PU.1 knockdown in vitro . (A) For the Aβ peptide phagocytosis assay, the cells were incubated alone with Hilyte-555-labeled fAβ (1–42) for 1 h (1.0 μM). After viral transduction, both TREM2 and PU.1 knockdown reduced microglial phagocytosis of fAβ (1–42). The mean fluorescence intensity (B) was quantified and is presented as the mean ± S.E.M. (C) For the microsphere phagocytosis assay, the cells were treated with Nile red microspheres (red) for 30 min. The cells were stained with phalloidin to visualize F-actin (green), and the percentages of microglia that underwent phagocytosis (C) and phagocytic efficiency (D) are presented as the means ± S.E.M.s (** p < 0.01). Scale bar = 10 μm. The data were quantified relative to those of the untreated group and analyzed via ANOVA with Bonferroni post hoc correction. * p < 0.05, *** p < 0.001, knockdown group versus control virus group.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: TREM2 (sheep anti-TREM2, R&D Systems) and PU.1 (1:300, rabbit anti-PU.1, Cell Signaling).

Techniques: Knockdown, In Vitro, Phagocytosis Assay, Incubation, Labeling, Transduction, Fluorescence, Staining, Control, Virus

Journal: Life science alliance

Article Title: The late-onset Alzheimer's disease risk factor RHBDF2 is a modifier of microglial TREM2 proteolysis.

doi: 10.26508/lsa.202403080

Figure Lengend Snippet: Figure 1. Loss of iRhom2 blocks ADAM17 activity in microglia. (A) Scheme of the role of iRhom2/ADAM17 in ectodomain shedding. ADAM17 requires iRhom2 for substrate proteolysis of TREM2 at the surface of myeloid cells, resulting in release of soluble TREM2 (sTREM2). Signaling of TREM2 occurs through binding to DAP12 and subsequent phosphorylation of spleen tyrosine kinase (SYK). (B) Immunoblot analysis of BV2 cell lysates. Two different knockout (KO) lines were generated by single-clone expansion using CRISPR guide RNAs for both ADAM17 (A17) and iRhom2 (iR2). A non-targeting control guide RNA served as a control. Two biological replicates per single-cell clone are shown. Pro- and mature forms of ADAM17 are indicated with asterisks. To enhance separation of pro- and mature forms of ADAM17 (indicated), lysate proteins were deglycosylated with PNGase F. Calnexin served as a loading control. (C) Loss of ADAM17 protease activity in BV2 cells with a knockout of ADAM17 or iRhom2 (A17KO, iR2KO). Indicated BV2 KO cells were treated with LPS (100 ng/ ml) for 2 h. TNF was measured in conditioned medium by ELISA (N ≥4). Statistical analysis was performed using a two-way ANOVA with Tukey’s correction for multiple comparisons. (D) Immunoblot analysis of primary microglia (MG) isolated from WT or iRhom2−/−(iR2 KO) pups. Three biological replicates of either genotype are shown. To enhance separation of pro- and mature forms of ADAM17 (indicated), lysate proteins were deglycosylated with PNGase F. Calnexin served as a loading control. * Asterisks indicate non-specific bands. (E) Functional depletion of ADAM17 protease activity in iRhom2−/−(iR2KO) primary microglia. Primary microglia were treated or not with LPS (1 μg/ml) for 1 h. TNF was measured in the conditioned medium by ELISA (N ≥3). Statistical analysis was performed using a two-way ANOVA with Tukey’s correction for multiple comparisons. (C, E) Data information: data (C, E) are represented as means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (BAF1729; R&D Systems) at 0.125 μg/ ml for 90 min at RT.

Techniques: Activity Assay, Binding Assay, Phospho-proteomics, Western Blot, Knock-Out, Generated, CRISPR, Control, Enzyme-linked Immunosorbent Assay, Isolation, Functional Assay

Journal: Life science alliance

Article Title: The late-onset Alzheimer's disease risk factor RHBDF2 is a modifier of microglial TREM2 proteolysis.

doi: 10.26508/lsa.202403080

Figure Lengend Snippet: Figure 2. iRhom2 enables ADAM17 proteolysis of TREM2 in BV2 cells. (A, B) Proteomics hiSPECS analysis comparing BV2 iRhom2−/−(A) or ADAM17−/−(B) microglia with BV2 non-targeting control (NTC) microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 P-value for each protein (two-sample t test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, P = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than twofold in the log scale. Proteins with a P-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2−/−: KO #1 and #2) and ADAM17 (A17−/−: KO #1 and #2) clones were pooled and compared with the pooled secretome of the two NTC clones (NTC #1 and NTC #2). (C) Peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (BAF1729; R&D Systems) at 0.125 μg/ ml for 90 min at RT.

Techniques: Control, Clone Assay

Journal: Life science alliance

Article Title: The late-onset Alzheimer's disease risk factor RHBDF2 is a modifier of microglial TREM2 proteolysis.

doi: 10.26508/lsa.202403080

Figure Lengend Snippet: Figure 3. iRhom2 controls TREM2 proteolysis in primary microglia. (A) Proteomics hiSPECS analysis comparing the secretome of primary microglia isolated from WT or iRhom2−/−(iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the P-value (−log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, P = 0.05, s0 = 0.1). Proteins with a P-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than twofold in the log scale. (B) Peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using Quantitative Analysis of Regulated Intramembrane Proteolysis (see Fig 2C). (C) Validation of iRhom2/ADAM17- mediated TREM2 ectodomain shedding in primary iRhom2−/−microglia. Supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥7). A two-sided independent t test was performed. (D) Immunoblot analysis of TREM2 levels in primary microglial lysates derived from WT or iRhom2−/−pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as a loading control. Data information: data (C) are represented as means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (BAF1729; R&D Systems) at 0.125 μg/ ml for 90 min at RT.

Techniques: Isolation, Quantitative Proteomics, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: Life science alliance

Article Title: The late-onset Alzheimer's disease risk factor RHBDF2 is a modifier of microglial TREM2 proteolysis.

doi: 10.26508/lsa.202403080

Figure Lengend Snippet: Figure 4. iRhom2 deficiency enhances TREM2 signaling in myeloid cells. (A) Immunoblot analysis of TREM2 signaling in cell lysates isolated from WT or iRhom2−/−(iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, or TREM2, or antibodies against spleen tyrosine kinase (SYK) or phospho-SYK-Y519/520. Calnexin and actin served as a loading control. (B) Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2−/−

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (BAF1729; R&D Systems) at 0.125 μg/ ml for 90 min at RT.

Techniques: Western Blot, Isolation, Control

Journal: Nature cardiovascular research

Article Title: TREM2 protects from atherosclerosis by limiting necrotic core formation.

doi: 10.1038/s44161-024-00429-9

Figure Lengend Snippet: Fig. 1 | Trem2 expression patterns at the single-cell level in mouse and human atherosclerosis. a, Uniform manifold approximation and projection (UMAP) visualization of scRNA-seq profiles of total mouse aortic cells in Ldlr−/− mice fed normal chow or an HFD for 8 weeks, 16 weeks or 26 weeks (Ldlr−/− HFD), n = 1 scRNA-seq library per timepoint. b, Expression of Trem2 in murine aortas projected onto the UMAP plot, split according to experimental condition. c, UMAP plot of mouse aortic MPCs identified in a after subsetting and reclustering. Inflamm., inflammatory; Mono, monocyte; (p)DC, (plasmacytoid) dendritic cell; Prolif., proliferating. d, Proportion of MPC clusters among all MPCs, and macrophage clusters among all macrophages, at different times of HFD feeding. e, Trem2 expression levels in MPC clusters. UMAP visualization of scRNA-seq of human atherosclerotic coronary artery cells (n = 4 patients, data from ref. 9) (f) and expression of TREM2 projected onto the UMAP plot (g). UMAP visualization of scRNA-seq profiles of human coronary artery MPCs

Article Snippet: The plate was then incubated for 90 min at room temperature with 0.125 μg ml−1 biotinylated polyclonal goat anti-mouse TREM2 capture antibody (R&D Systems, BAF1729) diluted in blocking buffer.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAP visualization of mouse hippocampal cell clusters classified by cell type based on DEG identified by Seurat v4. Violin plots represent the log-normalized expression of Itm2b and Trem2 across cell populations in mouse hippocampal cell clusters. ( B ) Human DFC cell clusters, classified by cell type as in ( A ). Violin plots represent the log-normalized expression of ITM2B and TREM2 across cell populations in human DFC cell clusters. ( C ) List of cell-type- specific marker genes used to annotate major brain populations. ( D ) Itm2b and Trem2 mRNA expression in mouse microglia and non-microglia cells analyzed by quantitative RT-PCR. Data information: The data sets analyzed are publicly available and are described in the two following papers: mouse scRNAseq data set (Zeisel et al, ), hippocampus n = 5 females, n = 5 males; human snRNAseq data set (Li et al, ), n = 10 (sex not specified). More information about these datasets can be found in the cited paper. Statistical comparisons between the groups shown in ( D ) was conducted using two-tailed unpaired t test **** P < 0.0001. The data are derived from are from 15-month-old w/w control animal, male n = 3, females n = 3; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Marker, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Control

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of objects Data 1 and Data 2 before filtering. ( B ) UMAPs of objects Data 1 and Data 2 after filtering. ( C ) UMAP of Object 1 after integration, filtering, and re-clustering, shows 16 microglia clusters. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. These cells derive from: Trem2-KO , 1 male and 1 female; Itm2b-KO , 2 males and 2 females; WT controls, 1 male and 2 females; Itm2b/Trem2-dKO , 1 male and 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques:

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of microglia grouped by genotype. ( B ) Average scaled expression levels of selected signature genes per cluster and cluster’s annotation based on expression of signature genes. ( C ) Volcano plots showing differentially expressed genes in clusters I/T-D1, 2, 3 and 4. ( D ) Proportional contribution of each genotype and proportional contribution of individual samples of each genotype to cluster 3. ( E ) KEGG pathway enrichment analysis of pathways upregulated in cluster 3. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. Volcano plots in ( C ) were obtained using Fast Wilcoxon rank sum test and auROC. These cells derive from: Trem2-KO , n = 1 male and n = 1 female; Itm2b-KO , n = 2 males and n = 2 females; WT controls, n = 1 male and n = 2 females; Itm2b/Trem2-dKO , n = 1 male and n = 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published. All data are expressed as means +/− SEM.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) N2A or HEK293 cells were transfected with F-BRI2 (B) and Trem2 (T), either alone (V=empty pcDNA3.1vector) or in combination and analyzed by Western blot with anti-FLAG (M2) and anti-Trem2 (NT1) on total lysates (T.L.) and M2 immunoprecipitants (IP-M2). Immunoprecipitants bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. For each cell line, two independent transfections were performed (Exp. 1 and Exp. 2). ( B ) Schematic representation of Trem2 and the two products of α-secretase cleavage, sTrem2, and Trem2-CTF. TM indicates the transmembrane region of Trem2. Red bars point to the antigenic regions used to produce the anti-Trem2 antibodies CT, NT1 and NT2. The cytosolic and intralumenal/extracellular regions of Trem2 are indicated. ( C ) Western blot analysis with anti-FLAG, anti-Trem2 NT1, and anti-Trem2 CT antibodies of T.L. and IP-M2 from HEK293 cells transfected with F-BRI2 and Trem2, either alone or in combination, with or without deglycosylation. *Indicates Trem2 species of unclear primary structure. Trem2 (f.l.) indicates full length Trem2. ( D ) Western blot analysis with anti-FLAG and anti-Trem2 CT antibodies of immunoprecipitants obtained with CT, NT1, and NT2 antibodies from HEK293 cells expressing either F-BRI2 alone or F-BRI2 plus Trem2. The nature of the bands migrating above 100 kDa in the NT2 IP samples is unknow. ( E ) Schematic representation of the F-BRI2 constructs used in ( F ). The Bri23 region, transmembrane region (TM), Brichos domain, APP-binding domain (APP BD), FLAG tag (F), cytosolic and intralumenal/extracellular regions are indicated. ( F ) WB analysis with anti-FLAG and anti-Trem2 antibodies of lysates and immunoprecipitants from HEK293 cells expressing F-BRI2 deletion mutants plus Trem2 or Trem2 alone (V). The * indicates Trem2 species of unclear primary structure. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Western Blot, Expressing, Construct, Binding Assay, FLAG-tag

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of the bicistronic expression plasmids used for HEK293 cell transfections in Panel ( B ): TREM2-3xFLAG + Myc-BRI2, 3xFLAG + Myc-BRI2 1-131 , 3xFLAG + Myc-BRI2 1-80 , and 3xFLAG + Myc-BRI2δ 80-131 . The TREM2-3xFLAG is expressed by the 5’ cistron, while BRI2 proteins are expressed by the 3’ cistron. ( B ) Western blot analysis using anti-FLAG, anti-Myc, and anti-human BRI2 antibodies of Total Lysate and Immunoprecipitation (M2-IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. The anti-human BRI2 antibody exhibits reactivity toward Myc-BRI2 and Myc-BRI2 1-131 suggesting recognition of an epitope located within the amino acids 80-131 region of BRI2. ( C ) Schematic representation of the bicistronic expression plasmids employed for HEK293 cell transfections in Panel ( D ): 3xFLAG-BRI2 + TREM2-Myc, 3xFLAG-BRI2 + TREM2-δIg-like-Myc, 3xFLAG-BRI2 + TREM2-CTF-Myc, 3xFLAG-BRI2 + TREM2-W198Ter-Myc, and 3xFLAG-BRI2 + TREM2-δ/α-site-Myc. The 3xFLAG-BRI2 is expressed by the 5’ cistron, while TREM2 proteins are expressed by the 3’ cistron. ( D ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of Total Lysate (T.L.) and Immunoprecipitation (IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. * Indicates protein signals of unclear nature. ** and *** indicate TREM2W198Ter signals of unclear primary structure. A longer exposure (Long Exposure) of the Anti-hTREM2-CT Western blot for the 3xFLAG-BRI2 + TREM2-Myc transfection revealed traces of TREM2-CTF precipitating with BRI2. Data information: This figure represents one of three independent experiments conducted. Data from the other two experiments are presented in Fig. . We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with anti-FLAG, anti-Myc antibodies, and anti-human BRI2 antibodies of total lysates and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( B ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of total lysates (T.L.) and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( C ) Co-immunoprecipitation of endogenous Bri2 and Trem2 from mouse primary macrophages. Samples were deglycosylated before Western blot. Data Information: Panels ( A ) and ( B ) represent two independent experiments conducted similarly to those in Figs. B and , respectively. Panel ( C ) shows the only co-immunoprecipitation of endogenous Bri2 and Trem2 performed to date. The complete membrane images used for Western blot analyses are included without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Immunoprecipitation, Transfection, Membrane

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of TREM2-ECD, sTREM2, BRI2-ECD and BRI2-BRICHOS recombinant proteins. TREM2-ECD encompasses the entire extracellular domain of TREM2, and BRI2-ECD encompasses the entire extracellular domain of BRI2, including the second putative TREM2-interacting domain. BRI2-BRICHOS and BRI2-ECD were fused with a 3xFLAG tag at their N-terminus, enabling immunoprecipitation using anti-FLAG M2-Agarose beads for the purification of protein complexes in a cell-free system via elution with a 3xFLAG peptide. The diagram highlights the signal peptides (SP), 7-Histidine tag (7-His, employed for protein purification), the 3XFLAG tag (3xF, utilized for complex purification), Ig-like domain (of TREM2), and BRICHOS domain (of BRI2). ( B ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated overnight at 4 degrees Celsius with M2-Agarose beads at a concentration of 2 μM for each protein. Following extensive washing, complexes bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. Unbound proteins and eluates (3xFLAG elu.) were analyzed by Western blot using either the anti-FLAG antibody M2 or an anti-human TREM2 N-terminal antibody (TREM2-NT). sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( C ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, 3xFLAG + sTREM2, 3xFLAG + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated as in B. Proteins eluted with the 3xFLAG peptide were analyzed by Western blot using either M2 or TREM2-NT. sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( D ) Western blot analysis using M2 and TREM2-NT antibodies of a new experiment mirroring the setup in ( B ). Eluates were separated under reducing and non-reducing conditions. BRI2-BRICHOS and BRI2-ECD monomers, dimers, trimers, and tetramers are indicated by the numbers 1, 2, 3, and 4, respectively. Higher multimolecular complexes are present but not labeled. The sTREM2 and TREM2-ECD bound to BRI2-BRICHOS and BRI2-ECD analyzed under non-reducing conditions show no significant increase in molecular weight compared to those analyzed under reducing conditions. ( E ) Decreasing concentrations (4, 2, 1, 0.5, 0.25, and 0 μM) of BRI2-BRICHOS and BRI2-ECD were incubated with 2 μM of either sTREM2 or TREM2-ECD and analyzed as described in ( C ). The bottom panel displays a Western blot of deglycosylated eluates using the TREM2-NT antibody. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Immunoprecipitation, Purification, Protein Purification, Incubation, Concentration Assay, Western Blot, Labeling, Molecular Weight

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) HEK293 cells were transfected with Trem2 and either empty vector (V) or F-BRI2 (B). NT are non-transfected cells. Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1. *Indicates Trem2 species of unclear primary structure. ( B ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( A ). ( C ) HEK293 cells were transfected with Trem2 and either empty vector (V), F-BRI2 or deletion mutant F-BRI2 1-80 . Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1 (lower panel). The * indicates Trem2 species of unclear primary structure. ( D ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( C ). ( E ) HEK293 cells were transfected with either Trem2 (T) or empty vector (V). Following transfection, lysates and media underwent deglycosylation and were subsequently analyzed by Western blot using anti-Trem2 antibodies CT and NT1. In the CT Western blot, the asterisk (*) indicates a Trem2-derived polypeptide that retains the CT epitope and is likely to lack part of the N-terminal Trem2 sequence, causing a reduction in size. In the NT1 Western blot, the double asterisk (**) highlights a Trem2-derived polypeptide that retains the NT1 epitope and is likely to lack part of the C-terminal sequence. The presence of this band primarily in cell lysates suggests potential retention of the transmembrane region and/or localization within intracellular compartments. Its low-level detection in the media further suggests intracellular origin. The band marked as sTrem2 is marked as sTrem2 because: (1) is of the expected size for deglycosilated sTrem2; (2) it is notably enriched in the media, consistent with the preferential localization of sTrem2 in extracellular fluids. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using two-tailed unpaired t test ( B ) and one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( C , D ). * P < 0.05, ** P < 0.01, *** P < 0.001. The data presented are derived from are from: Trem2+Vector transfectant n = 5, Trem2+F-BRI2 transfectant n = 5 ( A , B ); Trem2+Vector transfectant n = 3, Trem2+F-BRI2 transfectant n = 3, Trem2+F-BRI2 1-80 n = 3 ( C , D ); the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Derivative Assay, Sequencing, Two Tailed Test

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of ELISA 1 and ELISA 2. Both ELISAs use the same Biotinylated-αTrem2 capture antibody (in black). ELISA 1 uses αTrem2-CT (red) + Sulfo-αRabbit (blue) detection antibodies. ELISA 2 uses αTrem2-NT (orange) + Sulfo-αRat (green) detection antibodies. Trem2 can be detected by both ELISAs, sTrem2 can be detected only by ELISA 2: neither ELISA can detect Trem2-CTF. ( B ) Quantification of Trem2 and sTrem2 in the P100 and S100 brain fractions of ~245 days old w/w control, Itm2b-KO and Trem2-KO mice. ( C ) Western blot analysis of P100 fractions from a representative w/w, Trem2-KO and Itm2b-KO P100 sample with αTrem2-CT and an αBri2 antibody. *Indicates a non-specific band. ( D ) Detection and quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software; GAPDH was used as a loading control. ( E ) ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of w/w, Trem2-KO and Itm2b-KO animals. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. The membrane in ( C ) was cut at the 20 and 15 kDa molecular weight marker (MWM). The upper section was probed with the anti-Bri2 antibody, while the lower section was probed with the Trem2-CT antibody. Similarly, in ( D ), the two membranes were divided at the 20 and 15 kDa MWM. The upper portion was probed with the anti-Gapdh antibody, while the lower portion was probed with the Trem2-CT antibody. Statistical comparisons among the groups were conducted using one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( B , E ); two–way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( D ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from are from w/w control, females n = 7, males n = 12; Itm2b-KO females n = 6, males n = 7; Trem2-KO , females n = 6, males n = 7; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Software, Membrane, Molecular Weight, Marker, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) CD11b and CD45 staining, and FACS analysis of brain cells isolated from Cx3cr1 CreER/wt and Cx3cr1 wt/wt animals. ( B ) FACS analysis of sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from Itm2b f/f :Cx3cr1 CreER/wt animals. ( C ) Schematic representation of the PCR test used to identify the Itm2b f and Itm2b KO alleles. ( D ) PCR analysis of genomic DNA isolated from EYFP + and EYFP - cells sorted from Itm2b f/f :Cx3cr1 CreER/wt brains. ( E ) Analysis of Itm2b and Trem2 mRNA expression in sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from ~14 months-old Itm2b f/f :Cx3cr1 CreER/wt and Itm2b w/w :Cx3cr1 CreER/wt animals. ( F ) ELISA 2 was used to measure sTrem2 levels in Itm2b f/f :Cx3cr1 CreER/wt and Itm2b f/f :Cx3cr1 wt/wt littermates. Data information: Statistical comparisons among the groups were conducted two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( E ); two-tailed unpaired t test ( F ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from: (E) Itm2b f/f :Cx3cr1 CreER/wt , females n = 5, males n = 7; Itm2b w/w :Cx3cr1 CreER/wt , females n = 3, males n = 4; ( F ) Itm2b f/f :Cx3cr1 CreER/wt , females n = 15, males n = 12; Itm2b f/f :Cx3cr1 wt/wt , females n = 10, males n = 11; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the S100 brain fractions of ~245 days old control and FDD-KI mice. Data were analyzed by unpaired T -test. All data are shown as means +/− SEM: **** P < 0.0001. ( B ) Quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software. ( C ) Red Ponceau staining (upper panel) was used to normalize the Trem2-CTF signal (lover panel) obtained by Western blot. Data information: Statistical comparisons among the groups were conducted using unpaired T -test. **** P < 0.0001. The data presented are derived from: ( A ) w/w mice (females, n = 7; males, n = 12) and FDD-KI mice (females, n = 8; males, n = 9) mice; ( B ) w/w mice (females, n = 3; males, n = 3) and FDD-KI mice (females, n = 6; males, n = 7) mice; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Control, Western Blot, Software, Staining, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia using ELISA (left panel) and Western blot of deglycosylated conditioned media with Trem2 NT antibody (quantification of Western blot is shown in the second panel). Trem2 CT antibody does not show any signal. ( B ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 5 h of serum starvation by ELISA. ( C ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 1 and 2 h with either vehicle (Veh) or E. coli by ELISA. ( D ) Western blot of deglycosylated cell lysates from WT and Itm2b-KO primary microglia, 2 h after E. coli stimulation, with Trem2 CT antibody to visualize Trem2 f.l. and Trem2-CTF, along with quantification of the Trem2-CTF/Trem2 f.l. ratio. ( E ) Quantification of the Trem2-CTF/Trem2 f.l. ratio for only the two untreated (Veh) groups. ( F ) Analysis of Itm2b and Trem2 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( G ) A second set of biological replicates was analyzed following the same procedure as in panel ( C ). ( H ) A second set of biological replicates was analyzed following the same procedure as in panel ( D ). ( I ) A second set of biological replicates was analyzed following the same procedure as in panel ( F ). Data information: Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B , E , F , I ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C , D , G , H ). * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3 for ( A , C , D , E – I ), n = 6 for ( B )) and Itm2b-KO primary microglia ( n = 3 for Exp. and n = 3 for ( A , C , D , E – I ), n = 6 for ( B )); the letter “n” indicates biological replicates, except for ( B ), which includes 3 biological replicates with 2 technical replicates each. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with Trem2 CT antibody of deglycosylated cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the Trem2-CTF/Trem2 f.l. ratios from the Western blot shown in the right panel. ( B ) sTrem2 ELISA on conditioned media from these cell cultures (left panel). The right panel shows an ELISA performed using media from cells treated with vehicle and incubated before and during the ELISA with either vehicle (PBS) or 2 μM of BRI2-ECD. The evidence that incubation with BRI2-ECD does not change the ELISA quantification indicates that BRI2-ECD does not interfere with the quantification of sTrem2 by ELISA. ( C ) Western blot analysis with anti-Syk and anti-pSyk antibodies of cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the pSyk/Syk ratios from the Western blot is shown in the right panel. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using a two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001. The data presented are derived from Itm2b-KO primary microglia ( n = 3 for each condition); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction