anti human trem2  (R&D Systems)

 
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    Name:
    Human TREM 2 Antibody
    Description:
    The Human TREM 2 Antibody from R D Systems is a mouse monoclonal antibody to TREM 2 This antibody reacts with human The Human TREM 2 Antibody has been validated for the following applications Western Blot
    Catalog Number:
    MAB1828
    Price:
    289
    Category:
    Primary Antibodies
    Applications:
    Western Blot
    Purity:
    Protein A or G purified from hybridoma culture supernatant
    Conjugate:
    Unconjugated
    Immunogen:
    Mouse myeloma cell line NS0-derived recombinant human TREM-2, His19-Ser174, Accession # Q9NZC2
    Size:
    100 ug
    Host:
    Mouse
    Isotype:
    IgG2b
    Buy from Supplier


    Structured Review

    R&D Systems anti human trem2
    Exon 3 of <t>TREM2</t> is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .
    The Human TREM 2 Antibody from R D Systems is a mouse monoclonal antibody to TREM 2 This antibody reacts with human The Human TREM 2 Antibody has been validated for the following applications Western Blot
    https://www.bioz.com/result/anti human trem2/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human trem2 - by Bioz Stars, 2021-06
    93/100 stars

    Images

    1) Product Images from "Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation"

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25204-2

    Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Sequencing, Mutagenesis

    Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Modification, Sequencing, Construct, Splicing Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing

    Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.
    Figure Legend Snippet: Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

    Techniques Used: Mutagenesis

    Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Modification, Stable Transfection, Transfection, Western Blot, Expressing, Immunofluorescence, Staining, Construct

    Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

    NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Mutagenesis, Splicing Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing

    Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Expressing, Modification, Construct, Splicing Assay, Transfection, Western Blot

    2) Product Images from "Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation"

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25204-2

    Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 .
    Figure Legend Snippet: Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 .

    Techniques Used: Sequencing, Mutagenesis

    Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A .
    Figure Legend Snippet: Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A .

    Techniques Used: Modification, Sequencing, Construct, Splicing Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing

    Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.
    Figure Legend Snippet: Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

    Techniques Used: Mutagenesis

    Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F .
    Figure Legend Snippet: Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F .

    Techniques Used: Modification, Stable Transfection, Transfection, Western Blot, Expressing, Immunofluorescence, Staining

    Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t .
    Figure Legend Snippet: Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

    NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B .
    Figure Legend Snippet: NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B .

    Techniques Used: Mutagenesis, Splicing Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs .
    Figure Legend Snippet: Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs .

    Techniques Used: Expressing, Modification, Construct, Splicing Assay, Transfection, Western Blot

    3) Product Images from "Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation"

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25204-2

    Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Sequencing, Mutagenesis

    Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Modification, Sequencing, Construct, Splicing Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing

    Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.
    Figure Legend Snippet: Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

    Techniques Used: Mutagenesis

    Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Modification, Stable Transfection, Transfection, Western Blot, Expressing, Immunofluorescence, Staining, Construct

    Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

    NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .

    Techniques Used: Mutagenesis, Splicing Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing

    Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .
    Figure Legend Snippet: Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Techniques Used: Expressing, Modification, Construct, Splicing Assay, Transfection, Western Blot

    Related Articles

    Activation Assay:

    Article Title: Diabetic phenotype in mouse and humans with β-amyloid pathology reduces the number of microglia around β-amyloid plaques
    Article Snippet: .. Related to this, we now show that the ligand-induced activation of Trem2/Dap12 both directly (Trem2 antibody) and indirectly (M-CSF) upon the inhibition of PI3K-Akt signaling enhanced the activation of Syk kinase in mouse microglia. .. This was an unexpected result since it has been previously shown that the inhibition of PI3K in macrophages and osteoclasts prevented the recruitment of p85α and Syk kinase to Dap12, leading to reduced downstream signaling via other mediators, such as Plcγ2 [ ].

    Inhibition:

    Article Title: Diabetic phenotype in mouse and humans with β-amyloid pathology reduces the number of microglia around β-amyloid plaques
    Article Snippet: .. Related to this, we now show that the ligand-induced activation of Trem2/Dap12 both directly (Trem2 antibody) and indirectly (M-CSF) upon the inhibition of PI3K-Akt signaling enhanced the activation of Syk kinase in mouse microglia. .. This was an unexpected result since it has been previously shown that the inhibition of PI3K in macrophages and osteoclasts prevented the recruitment of p85α and Syk kinase to Dap12, leading to reduced downstream signaling via other mediators, such as Plcγ2 [ ].

    Staining:

    Article Title: TREM-2 promotes acquired cholesteatoma-induced bone destruction by modulating TLR4 signaling pathway and osteoclasts activation
    Article Snippet: After the slides were incubated in 10% H2 O2 in methanol for 10 minutes, endogenous peroxidase activity was blocked. .. Then, the slides were stained with an antibody directed against TREM-2 (human TREM-2 goat pAb, 1:10 dilution; R & D Systems) or its negative control IgG antibody (goat IgG, 1:10 dilution; Sigma) for no less than 8 hours at 4 °C. .. Then, the slides were incubated with a prepared polyclonal secondary antibody directed against human, sheep and mouse for 1 hour at 37 °C.

    Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism
    Article Snippet: A portion of cortex was preserved in RNAlater (Ambion) until all samples were collected. .. Confocal microscopy Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R & D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R & D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature. .. All antibodies were used in blocking buffer, and between all incubations, sections were washed for 10 min in PBS three times.

    Negative Control:

    Article Title: TREM-2 promotes acquired cholesteatoma-induced bone destruction by modulating TLR4 signaling pathway and osteoclasts activation
    Article Snippet: After the slides were incubated in 10% H2 O2 in methanol for 10 minutes, endogenous peroxidase activity was blocked. .. Then, the slides were stained with an antibody directed against TREM-2 (human TREM-2 goat pAb, 1:10 dilution; R & D Systems) or its negative control IgG antibody (goat IgG, 1:10 dilution; Sigma) for no less than 8 hours at 4 °C. .. Then, the slides were incubated with a prepared polyclonal secondary antibody directed against human, sheep and mouse for 1 hour at 37 °C.

    Blocking Assay:

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation
    Article Snippet: Forty-eight hours after transfection, the cells were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100 for 5 min at room temperature. .. After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight. .. Next, the cells were incubated with Alexa568-conjugated donkey anti-goat secondary antibody (1:1000; Thermo Fisher scientific) for 1 h at room temperature.

    Incubation:

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation
    Article Snippet: Forty-eight hours after transfection, the cells were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100 for 5 min at room temperature. .. After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight. .. Next, the cells were incubated with Alexa568-conjugated donkey anti-goat secondary antibody (1:1000; Thermo Fisher scientific) for 1 h at room temperature.

    Article Title: TREM-2 Binds to Lipooligosaccharides of Neisseria gonorrhoeae and is Expressed on Reproductive Tract Epithelial Cells
    Article Snippet: After removal of unbound bacteria by washing, bacterial binding to cells was detected by analyzing cells for associated fluorescence using a FACScan flow cytometer. .. For flow cytometric analyses of the membrane expression of human TREM-2 by all cell lines except HeLa, 5×105 cells were incubated for 60 min on ice with 1.5 μg/ml of anti-human TREM-2 MAb (R & D Systems, Minneapolis, MN) in a total volume of 100 μl of PBS containing 0.1% BSA. .. After 2 washes with PBS/BSA, the cells were incubated for 30 min on ice with 1 μg of FITC-conjugated rat anti-mouse IgG (eBioscience, San Diego, CA) in a total volume of 100 μl of PBS containing 0.1% BSA.

    Flow Cytometry:

    Article Title: TREM-2 Binds to Lipooligosaccharides of Neisseria gonorrhoeae and is Expressed on Reproductive Tract Epithelial Cells
    Article Snippet: After removal of unbound bacteria by washing, bacterial binding to cells was detected by analyzing cells for associated fluorescence using a FACScan flow cytometer. .. For flow cytometric analyses of the membrane expression of human TREM-2 by all cell lines except HeLa, 5×105 cells were incubated for 60 min on ice with 1.5 μg/ml of anti-human TREM-2 MAb (R & D Systems, Minneapolis, MN) in a total volume of 100 μl of PBS containing 0.1% BSA. .. After 2 washes with PBS/BSA, the cells were incubated for 30 min on ice with 1 μg of FITC-conjugated rat anti-mouse IgG (eBioscience, San Diego, CA) in a total volume of 100 μl of PBS containing 0.1% BSA.

    Expressing:

    Article Title: TREM-2 Binds to Lipooligosaccharides of Neisseria gonorrhoeae and is Expressed on Reproductive Tract Epithelial Cells
    Article Snippet: After removal of unbound bacteria by washing, bacterial binding to cells was detected by analyzing cells for associated fluorescence using a FACScan flow cytometer. .. For flow cytometric analyses of the membrane expression of human TREM-2 by all cell lines except HeLa, 5×105 cells were incubated for 60 min on ice with 1.5 μg/ml of anti-human TREM-2 MAb (R & D Systems, Minneapolis, MN) in a total volume of 100 μl of PBS containing 0.1% BSA. .. After 2 washes with PBS/BSA, the cells were incubated for 30 min on ice with 1 μg of FITC-conjugated rat anti-mouse IgG (eBioscience, San Diego, CA) in a total volume of 100 μl of PBS containing 0.1% BSA.

    Confocal Microscopy:

    Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism
    Article Snippet: A portion of cortex was preserved in RNAlater (Ambion) until all samples were collected. .. Confocal microscopy Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R & D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R & D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature. .. All antibodies were used in blocking buffer, and between all incubations, sections were washed for 10 min in PBS three times.

    Plasmid Preparation:

    Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism
    Article Snippet: A portion of cortex was preserved in RNAlater (Ambion) until all samples were collected. .. Confocal microscopy Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R & D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R & D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature. .. All antibodies were used in blocking buffer, and between all incubations, sections were washed for 10 min in PBS three times.

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    R&D Systems anti human trem2 antibody
    <t>TREM2-CLuc-IRES-TYROBP-NLuc</t> split- Renilla luciferase reporter system. The TREM2-CLuc-IRES-TYROBP-NLuc vector contains CMV immediate early ( IE ) promoter, the C terminus of the Renilla luciferase gene fused to the cytoplasmic region of TREM2 (TREM2-CLuc), IRES, and the N-terminal region of the Renilla luciferase gene fused to the N-terminal region of TYROBP (TYROBP-NLuc) ( A ). The unique restriction sites for cloning are depicted. Amp r , ampicillin resistance gene; Neo r , neomycin resistance gene. In a resting state, wild-type TREM2 and TYROBP show limited coupling. Upon stimulation with agonistic ligand (such as dimerizing anti-TREM2 antibody), TREM2 interacts with TYROBP, which reconstitutes luciferase activity by coupling of the C- and N-terminal regions of the split- Renilla luciferase gene ( B ).
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    Endogenous <t>TREM2</t> partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.
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    Expression of Th1-/Th2-type cytokines after in vivo silencing of <t>TREM-2.</t> ( A – F ) Real-time PCR data demonstrated that at 1 and 5 days p.i., silencing of TREM-2 upregulated the expression of Th1-type cytokines, including IFN-γ, AIL-12 ( B
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    R&D Systems anti human trem2
    Exon 3 of <t>TREM2</t> is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .
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    TREM2-CLuc-IRES-TYROBP-NLuc split- Renilla luciferase reporter system. The TREM2-CLuc-IRES-TYROBP-NLuc vector contains CMV immediate early ( IE ) promoter, the C terminus of the Renilla luciferase gene fused to the cytoplasmic region of TREM2 (TREM2-CLuc), IRES, and the N-terminal region of the Renilla luciferase gene fused to the N-terminal region of TYROBP (TYROBP-NLuc) ( A ). The unique restriction sites for cloning are depicted. Amp r , ampicillin resistance gene; Neo r , neomycin resistance gene. In a resting state, wild-type TREM2 and TYROBP show limited coupling. Upon stimulation with agonistic ligand (such as dimerizing anti-TREM2 antibody), TREM2 interacts with TYROBP, which reconstitutes luciferase activity by coupling of the C- and N-terminal regions of the split- Renilla luciferase gene ( B ).

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

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

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

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

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

    Techniques: Software

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

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

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

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

    Quantification of TREM2·TYROBP complex by “sandwich” ELISA. HEK293 cells were transfected with TREM2-CLuc-IRES-TYROBP-NLuc followed by anti-TREM2 antibody ( Ab ) stimulation to induce TREM2 coupling to TYROBP and protein cross-linking with DTBP and phosphatase inhibitor sodium pervanadate (Na 3 O 4 V) ( A ). Following cell lysis, protein was incubated in anti-TYROBP-coated wells and labeled for TREM2 with biotinylated anti-TREM2·HRP-conjugated streptavidin complex for quantification of TREM2·TYROBP protein complexes. Average ( Ave ) OD was quantified by ELISA. Data represent the arithmetic difference between a given treatment condition and the average OD of untransfected controls ( n = 4 per group) ( B ). Significant differences were determined by one-way ANOVA followed by Tukey's post-test. Error bars represent S.D. †† and ††† denote p

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

    Figure Lengend Snippet: Quantification of TREM2·TYROBP complex by “sandwich” ELISA. HEK293 cells were transfected with TREM2-CLuc-IRES-TYROBP-NLuc followed by anti-TREM2 antibody ( Ab ) stimulation to induce TREM2 coupling to TYROBP and protein cross-linking with DTBP and phosphatase inhibitor sodium pervanadate (Na 3 O 4 V) ( A ). Following cell lysis, protein was incubated in anti-TYROBP-coated wells and labeled for TREM2 with biotinylated anti-TREM2·HRP-conjugated streptavidin complex for quantification of TREM2·TYROBP protein complexes. Average ( Ave ) OD was quantified by ELISA. Data represent the arithmetic difference between a given treatment condition and the average OD of untransfected controls ( n = 4 per group) ( B ). Significant differences were determined by one-way ANOVA followed by Tukey's post-test. Error bars represent S.D. †† and ††† denote p

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

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

    Anti-TREM2 antibody, but not Aβ or anti-TYROBP antibody ( Ab ), induces dose-dependent increase in the TREM2-TYROBP interaction in HEK293 cells. HEK293 cells were transfected with internal control reporter pRL-TK, TREM2-CLuc-TYROBP negative control, or our TREM2-CLuc-IRES-TYROBP-NLuc plasmid prior to stimulation with ligand. Time-course data ( A , C , E , and G ) were assessed for luminescence over time by AUC analysis ( B , D , F , and H ) in RLU for n = 3 for each group. Error bars represent S.D. ααα denotes p

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

    Figure Lengend Snippet: Anti-TREM2 antibody, but not Aβ or anti-TYROBP antibody ( Ab ), induces dose-dependent increase in the TREM2-TYROBP interaction in HEK293 cells. HEK293 cells were transfected with internal control reporter pRL-TK, TREM2-CLuc-TYROBP negative control, or our TREM2-CLuc-IRES-TYROBP-NLuc plasmid prior to stimulation with ligand. Time-course data ( A , C , E , and G ) were assessed for luminescence over time by AUC analysis ( B , D , F , and H ) in RLU for n = 3 for each group. Error bars represent S.D. ααα denotes p

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

    Techniques: Transfection, Negative Control, Plasmid Preparation

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.759159

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

    Article Snippet: Cells were stimulated with 10, 30, or 100 μg/ml Aβ42; 1, 5, 10, or 20 μg/ml anti-human TREM2 antibody (rat monoclonal IgG2b clone 237920, MAB17291, R & D Systems, Minneapolis, MN); 1, 5, 10, or 20 μg/ml control rat IgG2b (eBioscience/Thermo Fisher Scientific, Inc.); or 5, 10, or 20 μg/ml anti-TYROBP antibody (rabbit polyclonal IgG, sc-20783, Santa Cruz Biotechnology, Dallas, TX) 24 h after transfection.

    Techniques: Mutagenesis, Transfection, Plasmid Preparation

    Endogenous TREM2 partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Endogenous TREM2 partially colocalizes with endogenous presenilin 1 (PS1) in microglial BV2 cells. BV2 cells were immunostained with antibodies against PS1 and mouse TREM2. White circles in magnified images indicate some colocalizing overlap between TREM2 and PS1 in Golgi-like structures. Scale bar, 5 μm.

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques:

    Upregulation of presenilin 1 (PS1) reduces steady-state levels of TREM2 at the cell surface. ( a ) Following PS1 overexpression, HEK293-TREM2 cells were treated with or without the γ-secretase inhibitor Compound E (CpdE) and subjected to cell surface biotinylation assay. Precipitates from streptavidin-agarose beads were immunoblotted for biotinylated TREM2 and total TREM2 levels (levels of TREM2 in 2% total cell lysates). * P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Upregulation of presenilin 1 (PS1) reduces steady-state levels of TREM2 at the cell surface. ( a ) Following PS1 overexpression, HEK293-TREM2 cells were treated with or without the γ-secretase inhibitor Compound E (CpdE) and subjected to cell surface biotinylation assay. Precipitates from streptavidin-agarose beads were immunoblotted for biotinylated TREM2 and total TREM2 levels (levels of TREM2 in 2% total cell lysates). * P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Over Expression, Cell Surface Biotinylation Assay

    Overexpression of presenilin 1 (PS1) impairs TREM2-mediated phagocytosis in microglial cells. ( a ) PS1 and mCherry were co-transfected into BV2 microglial cells. The cells were then incubated with 6-carboxyfluorescein (FAM)-labeled Aβ42 for 2 h. FAM-Aβ42 uptake was analyzed by fluorescence microscopy. Scale bars, 10 μm. ( b ) Phagocytosis of FAM-Aβ42 in BV2 cells stably expressing PS1 as determined by flow cytometry. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Overexpression of presenilin 1 (PS1) impairs TREM2-mediated phagocytosis in microglial cells. ( a ) PS1 and mCherry were co-transfected into BV2 microglial cells. The cells were then incubated with 6-carboxyfluorescein (FAM)-labeled Aβ42 for 2 h. FAM-Aβ42 uptake was analyzed by fluorescence microscopy. Scale bars, 10 μm. ( b ) Phagocytosis of FAM-Aβ42 in BV2 cells stably expressing PS1 as determined by flow cytometry. * P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

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

    Presenilin 1 (PS1) interacts with TREM2. ( a , b ) PS1 constructs were transfected into HEK293 cells stably expressing TREM2 with a Myc-tag at the C terminus (HEK293-TREM2). ( a ) Cell lysates were immunoprecipitated with a Myc antibody or control IgG. Immunoprecipitated proteins were subjected to immunoblotting with an Ab14 antibody to detect full-length PS1 (PS1-FL) and the PS1 N-terminal fragment (NTF), an anti-PS1 loop antibody to detect the PS1 C-terminal fragment (CTF) and a nicastrin (NCT) antibody as indicated. ( b ) Cell lysates were immunoprecipitated with Ab14, anti-PS1 loop or control IgG and immunoblotted with Myc and NCT antibodies. ( c ) Lysates from BV2 microglial cells were immunoprecipitated with Ab14 and immunoblotted with NCT and mouse TREM2 antibodies. ( d ) Vectors expressing wild-type (WT) or mutant PS1 (D385A) were transfected into HEK293-TREM2 cells. Cell lysates were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( e ) Lysates from HEK293-TREM2 cells with or without Compound E (CpdE, a γ-secretase inhibitor) treatment were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( f ) Schematic representations of full-length (1–230) or truncated TREM2 constructs, all tagged with GST at the C terminus. SP, signal peptide; TM, transmembrane domain. ( g ) PS1 was co-expressed with full-length TREM2 or other TREM2 fragments as shown in f in HEK293 cells. Cell lysates were precipitated with Glutathione Sepharose beads and immunoblotted with the PS1 antibody Ab14 or an antibody against GST. PS1 co-precipitation levels were determined by densitometric analysis and normalized with respect to both PS1 expression and precipitated GST. ** P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Presenilin 1 (PS1) interacts with TREM2. ( a , b ) PS1 constructs were transfected into HEK293 cells stably expressing TREM2 with a Myc-tag at the C terminus (HEK293-TREM2). ( a ) Cell lysates were immunoprecipitated with a Myc antibody or control IgG. Immunoprecipitated proteins were subjected to immunoblotting with an Ab14 antibody to detect full-length PS1 (PS1-FL) and the PS1 N-terminal fragment (NTF), an anti-PS1 loop antibody to detect the PS1 C-terminal fragment (CTF) and a nicastrin (NCT) antibody as indicated. ( b ) Cell lysates were immunoprecipitated with Ab14, anti-PS1 loop or control IgG and immunoblotted with Myc and NCT antibodies. ( c ) Lysates from BV2 microglial cells were immunoprecipitated with Ab14 and immunoblotted with NCT and mouse TREM2 antibodies. ( d ) Vectors expressing wild-type (WT) or mutant PS1 (D385A) were transfected into HEK293-TREM2 cells. Cell lysates were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( e ) Lysates from HEK293-TREM2 cells with or without Compound E (CpdE, a γ-secretase inhibitor) treatment were immunoprecipitated with Ab14 or control IgG, and PS1 was detected by immunoblotting. ( f ) Schematic representations of full-length (1–230) or truncated TREM2 constructs, all tagged with GST at the C terminus. SP, signal peptide; TM, transmembrane domain. ( g ) PS1 was co-expressed with full-length TREM2 or other TREM2 fragments as shown in f in HEK293 cells. Cell lysates were precipitated with Glutathione Sepharose beads and immunoblotted with the PS1 antibody Ab14 or an antibody against GST. PS1 co-precipitation levels were determined by densitometric analysis and normalized with respect to both PS1 expression and precipitated GST. ** P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Construct, Transfection, Stable Transfection, Expressing, Immunoprecipitation, Mutagenesis

    Mutations in TREM2 affect colocalization and interactions between TREM2 and presenilin 1 (PS1). PS1 was transfected into HEK293 cells stably expressing Myc-tagged TREM2 WT or TREM2 mutants as indicated. ( a , b ) Cells were then subjected to immunostaining with antibodies against Myc, PS1, and TGN46 (a marker for the Golgi, a ) or PDI (a marker for the ER, b ). White arrows in magnified images indicate colocalizing overlap for TREM2, PS1 and TGN46/PDI. Scale bars for a , b , 10 μm. ( c ) Quantification of colocalized signals. Pearson’s correlation coefficient is shown. *** P

    Journal: Experimental & Molecular Medicine

    Article Title: Intracellular trafficking of TREM2 is regulated by presenilin 1

    doi: 10.1038/emm.2017.200

    Figure Lengend Snippet: Mutations in TREM2 affect colocalization and interactions between TREM2 and presenilin 1 (PS1). PS1 was transfected into HEK293 cells stably expressing Myc-tagged TREM2 WT or TREM2 mutants as indicated. ( a , b ) Cells were then subjected to immunostaining with antibodies against Myc, PS1, and TGN46 (a marker for the Golgi, a ) or PDI (a marker for the ER, b ). White arrows in magnified images indicate colocalizing overlap for TREM2, PS1 and TGN46/PDI. Scale bars for a , b , 10 μm. ( c ) Quantification of colocalized signals. Pearson’s correlation coefficient is shown. *** P

    Article Snippet: For cell surface labeling experiments, cells were blocked in 5% BSA after fixation and stained with a goat anti-human TREM2 antibody (epitope 19–174 amino acids, R & D Systems).

    Techniques: Transfection, Stable Transfection, Expressing, Immunostaining, Marker

    Expression of Th1-/Th2-type cytokines after in vivo silencing of TREM-2. ( A – F ) Real-time PCR data demonstrated that at 1 and 5 days p.i., silencing of TREM-2 upregulated the expression of Th1-type cytokines, including IFN-γ, AIL-12 ( B

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: Expression of Th1-/Th2-type cytokines after in vivo silencing of TREM-2. ( A – F ) Real-time PCR data demonstrated that at 1 and 5 days p.i., silencing of TREM-2 upregulated the expression of Th1-type cytokines, including IFN-γ, AIL-12 ( B

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Expressing, In Vivo, Real-time Polymerase Chain Reaction

    Expression of TREM-2 in response to PA infection. ( A ) TREM-2 mRNA levels (relative gene expression after normalization to β-actin) were examined in normal uninfected and infected BALB/c corneas at 1, 3, and 5 days p.i. Data are the mean ±

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: Expression of TREM-2 in response to PA infection. ( A ) TREM-2 mRNA levels (relative gene expression after normalization to β-actin) were examined in normal uninfected and infected BALB/c corneas at 1, 3, and 5 days p.i. Data are the mean ±

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Expressing, Infection

    Expression of Th1/Th2 and proinflammatory cytokines after in vitro silencing of TREM-2. PCR data demonstrated that in LPS-stimulated RAW264.7 cells, silencing of TREM-2 increased expression of Th1 cytokines, such as IFN-γ, IL-12, and IL-18 ( A

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: Expression of Th1/Th2 and proinflammatory cytokines after in vitro silencing of TREM-2. PCR data demonstrated that in LPS-stimulated RAW264.7 cells, silencing of TREM-2 increased expression of Th1 cytokines, such as IFN-γ, IL-12, and IL-18 ( A

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Expressing, In Vitro, Polymerase Chain Reaction

    TREM-2 suppressed production of proinflammatory cytokines via PI3K/Akt signaling pathway. ( A ) Phosphorylated and total protein levels of Akt in RAW264.7 cells were examined by Western blot before and after treatment with agonistic TREM-2 antibody. Activation

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: TREM-2 suppressed production of proinflammatory cytokines via PI3K/Akt signaling pathway. ( A ) Phosphorylated and total protein levels of Akt in RAW264.7 cells were examined by Western blot before and after treatment with agonistic TREM-2 antibody. Activation

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Western Blot, Activation Assay

    Silencing of TREM-2 enhanced bacterial load and PMN infiltration after PA infection. Bacterial load ( A ) was enhanced in TREM-2 siRNA versus control-treated corneas at 5 days p.i. The number of infiltrated PMNs as detected by MPO assay ( B ) was elevated

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: Silencing of TREM-2 enhanced bacterial load and PMN infiltration after PA infection. Bacterial load ( A ) was enhanced in TREM-2 siRNA versus control-treated corneas at 5 days p.i. The number of infiltrated PMNs as detected by MPO assay ( B ) was elevated

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Infection, MPO Assay

    In vivo studies of TREM-2. ( A – G ) BALB/c mice were subconjunctivally injected with TREM-2 siRNA or scrambled control, and then infected with PA routinely. Clinical scores ( A ) indicated statistically significant differences at 3 and 5 days p.i.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: In vivo studies of TREM-2. ( A – G ) BALB/c mice were subconjunctivally injected with TREM-2 siRNA or scrambled control, and then infected with PA routinely. Clinical scores ( A ) indicated statistically significant differences at 3 and 5 days p.i.

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: In Vivo, Mouse Assay, Injection, Infection

    Expression of proinflammatory cytokines after in vivo silencing of TREM-2. Real-time PCR data demonstrated that at 1 and 5 days p.i., silencing of TREM-2 enhanced the mRNA levels of proinflammatory cytokines, including TNF-α ( A ), MIP-2 ( B ), and

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

    doi: 10.1167/iovs.12-10938

    Figure Lengend Snippet: Expression of proinflammatory cytokines after in vivo silencing of TREM-2. Real-time PCR data demonstrated that at 1 and 5 days p.i., silencing of TREM-2 enhanced the mRNA levels of proinflammatory cytokines, including TNF-α ( A ), MIP-2 ( B ), and

    Article Snippet: Primary antibodies (Abs) against TREM-2, phosphorylated Akt (p-Akt), Akt, and β-actin for Western blot were obtained from R & D Systems (Minneapolis, MN), Epitomics (Burlingame, CA), Cell Signaling Technology (Danvers, MA), and Sigma (St. Louis, MO), respectively.

    Techniques: Expressing, In Vivo, Real-time Polymerase Chain Reaction

    Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Sequencing, Mutagenesis

    Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Modification, Sequencing, Construct, Splicing Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing

    Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Mutagenesis

    Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Modification, Stable Transfection, Transfection, Western Blot, Expressing, Immunofluorescence, Staining, Construct

    Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

    NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Mutagenesis, Splicing Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing

    Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Journal: Scientific Reports

    Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

    doi: 10.1038/s41598-018-25204-2

    Figure Lengend Snippet: Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. S8 .

    Article Snippet: After blocking with 5% skim milk for at least 30 min, the cells were incubated with anti-human TREM2 (1:200) at 4 °C overnight.

    Techniques: Expressing, Modification, Construct, Splicing Assay, Transfection, Western Blot