sheep trem2  (R&D Systems)

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

Journal: bioRxiv

Article Title: Pharmacological PINK1 activation ameliorates Pathology in Parkinson’s Disease models

doi: 10.1101/2023.02.14.528378

Figure Lengend Snippet: (A) Related to . Immunostaining shows representative images of neurons treated with mPFFs and 2.8 µM MTK458. Primary hippocampal neurons were seeded for immunostaining, treated with mPFFs (0.5 µg/mL) and MTK458 as in (E), and on DIV14 fixed for immunostaining with antibodies against pS129 α-syn, NfH and TUJ1. Scale bar, 50 µm. (B) Mice were challenged with striatal injection of PFFs of PBS. After 2 weeks, ipsilateral or contralateral striatum brain pieces were harvested for sequential extraction with buffers containing increasing amounts of detergent. NP-40 insoluble fractions were analyzed by immunoblot. (C-D) Quantification of (B) is shown. (E-G) Mice were challenged with striatal injection of PBS or PFF, and then dosed with MTK458 for 6 months. Mice were analyzed for TREM2 in the ipsilateral striatum (E), plasma CXCL1 (F), and plasma IL-6 (G) levels. (H) Similar experiment to , except that mice were sacrificed 180 days after PFF injection and MTK458 dosing (QD, PO) at the indicated concentrations. (I-J) Quantification of (H) is shown. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001, n.s., not significant.

Article Snippet: Sheep anti-TREM2 polyclonal antibody (R&D systems BAF1729) was coated onto Small Spot Streptavidin 96 well plates (MSD, L45SA-2) as the capture antibody.

Techniques: Immunostaining, Injection, Extraction, Western Blot, Clinical Proteomics