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R&D Systems type ii nrg1
Type Ii Nrg1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 3 article reviews

type ii nrg1 - by Bioz Stars, 2026-06

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Generation of a mouse monoclonal antibody against Type II NRG1. A, Semiquantitative RT-PCR of NRG1 Types I, II, and III (CRD-NRG1 or SMDF) from E19 fetuses, P7 pups, and adult rat total brain RNA. Data were normalized to β-actin. Data points indicate mean ± SEM of three independent RNA samples, each run in duplicates. B, Schematic illustration of the generation of mouse monoclonal antibody 7C11 against the Type II NRG1 ECD (see Materials and Methods). C, Clone 7C11 selectively recognizes the NRG1 Type II ECD, as demonstrated by its reactivity toward Type II (middle), but not toward Type I NRG1 (top) in AAV-transduced hippocampal neurons, or endogenous NRG2 (bottom); MAP2 (white) was included in the overlay images to reveal neuronal morphology. Based on the lack of cross-reactivity of clone 7C11 with Type I NRG1, the 7C11 epitope is likely located in the Type II-specific sequence shown in green (B). Images are Z-stack projections. Scale bar, 10 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Generation of a mouse monoclonal antibody against Type II NRG1. A, Semiquantitative RT-PCR of NRG1 Types I, II, and III (CRD-NRG1 or SMDF) from E19 fetuses, P7 pups, and adult rat total brain RNA. Data were normalized to β-actin. Data points indicate mean ± SEM of three independent RNA samples, each run in duplicates. B, Schematic illustration of the generation of mouse monoclonal antibody 7C11 against the Type II NRG1 ECD (see Materials and Methods). C, Clone 7C11 selectively recognizes the NRG1 Type II ECD, as demonstrated by its reactivity toward Type II (middle), but not toward Type I NRG1 (top) in AAV-transduced hippocampal neurons, or endogenous NRG2 (bottom); MAP2 (white) was included in the overlay images to reveal neuronal morphology. Based on the lack of cross-reactivity of clone 7C11 with Type I NRG1, the 7C11 epitope is likely located in the Type II-specific sequence shown in green (B). Images are Z-stack projections. Scale bar, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Type I and Type II NRG1 accumulate as unprocessed proteins and are shed by matrix metalloproteinases in response to NMDAR activation by glutamate. A, B, Western blot of whole-cell lysates from cultured hippocampal neurons transduced with AAVs harboring V5-tagged Type I or Type II NRG1, and incubated with (A) anti-V5 to detect their unprocessed proforms and ECD fragments, or (B) SC-348 to detect unprocessed proforms and the processed ICD fragment. Both ECD and ICD antibodies reveal that the bulk of single-pass NRGs in cell lysates is unprocessed at steady state (Ctrl), consistent with the overlap between ECD and ICD signals in ICC (Fig. 2E) and IFH (Figs. 4F–J, ,5).5). Glutamate (Glu; 20 μm for 20 min) promotes the generation of ECD and ICD fragments. This effect is blocked by AP5 (+AP5), indicating that processing is regulated in an NMDA receptor-dependent manner. C, D, Heparin pulldown of the Type II NRG1 ectodomain from medium conditioned by AAV-transduced hippocampal neurons, using anti-V5 for Western blotting. Ectodomain shedding by glutamate is sensitive to inhibition of NMDA receptors (+AP5) and of metalloproteinases by GM6001 (10 μm; +GM) but not of BACE by BACE-IV (1 μm; +B-IV). D, Summary data were normalized to untreated controls (arbitrarily set to 1) and represent the mean ± SEM from four independent experiments. p values were derived from multiple pairwise comparisons between Glu and Glu + inhibitor treatments: *p < 0.05 (one-way ANOVA with Dunnett's post hoc test).

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Type I and Type II NRG1 accumulate as unprocessed proteins and are shed by matrix metalloproteinases in response to NMDAR activation by glutamate. A, B, Western blot of whole-cell lysates from cultured hippocampal neurons transduced with AAVs harboring V5-tagged Type I or Type II NRG1, and incubated with (A) anti-V5 to detect their unprocessed proforms and ECD fragments, or (B) SC-348 to detect unprocessed proforms and the processed ICD fragment. Both ECD and ICD antibodies reveal that the bulk of single-pass NRGs in cell lysates is unprocessed at steady state (Ctrl), consistent with the overlap between ECD and ICD signals in ICC (Fig. 2E) and IFH (Figs. 4F–J, ,5).5). Glutamate (Glu; 20 μm for 20 min) promotes the generation of ECD and ICD fragments. This effect is blocked by AP5 (+AP5), indicating that processing is regulated in an NMDA receptor-dependent manner. C, D, Heparin pulldown of the Type II NRG1 ectodomain from medium conditioned by AAV-transduced hippocampal neurons, using anti-V5 for Western blotting. Ectodomain shedding by glutamate is sensitive to inhibition of NMDA receptors (+AP5) and of metalloproteinases by GM6001 (10 μm; +GM) but not of BACE by BACE-IV (1 μm; +B-IV). D, Summary data were normalized to untreated controls (arbitrarily set to 1) and represent the mean ± SEM from four independent experiments. p values were derived from multiple pairwise comparisons between Glu and Glu + inhibitor treatments: *p < 0.05 (one-way ANOVA with Dunnett's post hoc test).

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Activation Assay, Western Blot, Cell Culture, Transduction, Incubation, Inhibition, Derivative Assay

$NRG3 accumulates on axonal varicosities, some of which are bona fide glutamatergic terminals. A, Hippocampal neurons were cotransfected with NRG3/V5 in pDESTDV3 and GFP. The representative low-magnification image shows GFP-positive processes with V5 signal accumulating at varicosities (arrowheads). B, Neurons were cotransfected with NRG3/V5, mCherry, and the axonal marker GFP-SNPH-ΔMTB. Left, The mCherry/V5 overlay image shows three NRG3 puncta on mCherry-positive processes (arrowheads). The presence of GFP-SNPH-ΔMTB signals in these processes in the corresponding mCherry/GFP-SNPH-ΔMTB overlay image (right) reveals that these processes are axons. C, Neurons cotransfected with CRD-NRG1/HA and NRG3/V5 show extensively overlapping immunoreactivity for both tags at axonal varicosities. D, E, CRD-NRG1 and NRG3 accumulate at presynaptic terminals. Top, Middle, Hippocampal neurons were transfected with NRG3/V5 (D) or CRD-NRG1/V5 (E) and colabeled with antibodies against V5, Bassoon, and PSD-95. The representative DIV 21 images illustrate the partial overlap between V5, the presynaptic marker Bassoon (top), and the postsynaptic marker PSD-95 (middle). Arrowheads indicate examples of synaptic sites that are positive for V5, Bassoon, and PSD-95. Bottom, Corresponding quantitative colocalization data for NRG3/V5 and CRD-NRG1/V5 derived from the analysis of V5 puncta at DIV 14, DIV 21, and DIV 28. For both NRG3 and CRD-NRG1, the fraction of V5 puncta colocalizing with synaptic markers significantly increases during in vitro development. Data are mean ± SEM of the fraction of V5 puncta colocalizing with synaptic markers from a total of 27–40 ROIs acquired in three independent experiments. p values were derived from multiple comparisons within each group using one-way ANOVA with Tukey's post hoc test. *p < 0.05. **p < 0.01. ***p < 0.001. Scale bars: A, 20 μm; B, C, 5 μm; D, E, 10 μm.$

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: NRG3 accumulates on axonal varicosities, some of which are bona fide glutamatergic terminals. A, Hippocampal neurons were cotransfected with NRG3/V5 in pDESTDV3 and GFP. The representative low-magnification image shows GFP-positive processes with V5 signal accumulating at varicosities (arrowheads). B, Neurons were cotransfected with NRG3/V5, mCherry, and the axonal marker GFP-SNPH-ΔMTB. Left, The mCherry/V5 overlay image shows three NRG3 puncta on mCherry-positive processes (arrowheads). The presence of GFP-SNPH-ΔMTB signals in these processes in the corresponding mCherry/GFP-SNPH-ΔMTB overlay image (right) reveals that these processes are axons. C, Neurons cotransfected with CRD-NRG1/HA and NRG3/V5 show extensively overlapping immunoreactivity for both tags at axonal varicosities. D, E, CRD-NRG1 and NRG3 accumulate at presynaptic terminals. Top, Middle, Hippocampal neurons were transfected with NRG3/V5 (D) or CRD-NRG1/V5 (E) and colabeled with antibodies against V5, Bassoon, and PSD-95. The representative DIV 21 images illustrate the partial overlap between V5, the presynaptic marker Bassoon (top), and the postsynaptic marker PSD-95 (middle). Arrowheads indicate examples of synaptic sites that are positive for V5, Bassoon, and PSD-95. Bottom, Corresponding quantitative colocalization data for NRG3/V5 and CRD-NRG1/V5 derived from the analysis of V5 puncta at DIV 14, DIV 21, and DIV 28. For both NRG3 and CRD-NRG1, the fraction of V5 puncta colocalizing with synaptic markers significantly increases during in vitro development. Data are mean ± SEM of the fraction of V5 puncta colocalizing with synaptic markers from a total of 27–40 ROIs acquired in three independent experiments. p values were derived from multiple comparisons within each group using one-way ANOVA with Tukey's post hoc test. *p < 0.05. **p < 0.01. ***p < 0.001. Scale bars: A, 20 μm; B, C, 5 μm; D, E, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Marker, Transfection, Derivative Assay, In Vitro

Ig-NRG1s accumulate on cell bodies and proximal dendrites in cultured hippocampal neurons. A, Schematic diagram of V5 epitope-tagged Type I and II NRG1β1a constructs used to analyze subcellular expression of Ig-NRG1s. B, Representative low-magnification confocal image of a neuron transduced with an AAV expressing Type I NRG1/V5, showing V5-immunoreactive puncta concentrated on the cell body and proximal MAP2-positive dendrites (yellow arrowheads), but not on distal dendrites or proximal axons identified by Ankyrin G staining (red arrowheads). C, Colocalization of endogenous NRG2 (mAB11) with Type I-NRG1/V5 (left) or Type II-NRG1/V5 (right) in AAV-transduced hippocampal neurons; neuronal morphology was revealed by MAP-2 staining. ROIs are shown at higher magnification (bottom). Arrowheads indicate examples of overlapping puncta. D, As previously reported for NRG2 (Vullhorst et al., 2015), NRG1 Type II puncta (arrowheads) are embedded in large clusters of Kv2.1 that likely correspond to SSCs. E, Extensively overlapping pattern of ECD and ICD signals for Type I NRG1, visualized with anti-V5 and the carboxyl-terminal antibody SC-348, indicate that Ig-NRG1 puncta are comprised of unprocessed protein. F, Immunogold EM images using antibody 7C11 against the ECD of Type II NRG1 (Fig. 3), showing label on the external side of the neuronal plasma membrane atop SSCs (open sacs indicated by arrows). Top, Axon without label in close contact and a highly concentrated patch of label immediately adjacent to the axon/soma contact area. Bottom, Labeled areas of the neuronal soma without any cellular processes in contact. G, The spatial relationship between Kv2.1 clusters and NRG2 puncta (arrowheads) is maintained in the absence of the Ig-like domain. Top, Schematic illustration of mutant NRG2 lacking the Ig-like domain (NRG2ΔIg/V5). Bottom, Double immunofluorescence of V5-tagged NRG2ΔIg and Kv2.1. Examples of coclusters are marked by arrowheads in the magnified single-channel ROI (right). All images are rendered as Z-stack projections. DAPI was included to label nuclei. Scale bars: main images, 10 μm; ROIs, 5 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Ig-NRG1s accumulate on cell bodies and proximal dendrites in cultured hippocampal neurons. A, Schematic diagram of V5 epitope-tagged Type I and II NRG1β1a constructs used to analyze subcellular expression of Ig-NRG1s. B, Representative low-magnification confocal image of a neuron transduced with an AAV expressing Type I NRG1/V5, showing V5-immunoreactive puncta concentrated on the cell body and proximal MAP2-positive dendrites (yellow arrowheads), but not on distal dendrites or proximal axons identified by Ankyrin G staining (red arrowheads). C, Colocalization of endogenous NRG2 (mAB11) with Type I-NRG1/V5 (left) or Type II-NRG1/V5 (right) in AAV-transduced hippocampal neurons; neuronal morphology was revealed by MAP-2 staining. ROIs are shown at higher magnification (bottom). Arrowheads indicate examples of overlapping puncta. D, As previously reported for NRG2 (Vullhorst et al., 2015), NRG1 Type II puncta (arrowheads) are embedded in large clusters of Kv2.1 that likely correspond to SSCs. E, Extensively overlapping pattern of ECD and ICD signals for Type I NRG1, visualized with anti-V5 and the carboxyl-terminal antibody SC-348, indicate that Ig-NRG1 puncta are comprised of unprocessed protein. F, Immunogold EM images using antibody 7C11 against the ECD of Type II NRG1 (Fig. 3), showing label on the external side of the neuronal plasma membrane atop SSCs (open sacs indicated by arrows). Top, Axon without label in close contact and a highly concentrated patch of label immediately adjacent to the axon/soma contact area. Bottom, Labeled areas of the neuronal soma without any cellular processes in contact. G, The spatial relationship between Kv2.1 clusters and NRG2 puncta (arrowheads) is maintained in the absence of the Ig-like domain. Top, Schematic illustration of mutant NRG2 lacking the Ig-like domain (NRG2ΔIg/V5). Bottom, Double immunofluorescence of V5-tagged NRG2ΔIg and Kv2.1. Examples of coclusters are marked by arrowheads in the magnified single-channel ROI (right). All images are rendered as Z-stack projections. DAPI was included to label nuclei. Scale bars: main images, 10 μm; ROIs, 5 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Cell Culture, Construct, Expressing, Transduction, Staining, Labeling, Mutagenesis, Immunofluorescence

Type II NRG1 mRNA and protein are expressed in GABAergic neurons of the RTN. A–E, Double-fluorescence ISH with RNAscope probes for Type II NRG1 and Gad1 in a representative coronal mouse section through the RTN. Low-magnification image showing overlays for both Type II NRG1 and Gad1 (A), or only Type II NRG1 (B), reveals strong Type II expression in numerous GABAergic neurons. A, Boxed ROI 1 is magnified in C, D. Arrowheads indicate examples of neurons coexpressing Type II NRG1 and Gad1 mRNA. Boxed ROI 2 outside the RTN is magnified in E to emphasize the lack of Gad1 and negligible NRG1 label. F–J, Triple immunofluorescence histochemistry of Type II NRG1 protein in adult mouse RTN neurons using antibodies against its ECD (7C11) and ICD (SC-348). F, Overlay shows numerous NRG puncta (colabeled with 7C11 and SC-348) on or near cell bodies of PV-positive GABAergic neurons. Boxed ROI is magnified in G–J to illustrate the extensive overlap between ECD and ICD signals (arrowheads). DAPI is included in all images to label nuclei. Scale bars: A, B, 200 μm; C–J, 10 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Type II NRG1 mRNA and protein are expressed in GABAergic neurons of the RTN. A–E, Double-fluorescence ISH with RNAscope probes for Type II NRG1 and Gad1 in a representative coronal mouse section through the RTN. Low-magnification image showing overlays for both Type II NRG1 and Gad1 (A), or only Type II NRG1 (B), reveals strong Type II expression in numerous GABAergic neurons. A, Boxed ROI 1 is magnified in C, D. Arrowheads indicate examples of neurons coexpressing Type II NRG1 and Gad1 mRNA. Boxed ROI 2 outside the RTN is magnified in E to emphasize the lack of Gad1 and negligible NRG1 label. F–J, Triple immunofluorescence histochemistry of Type II NRG1 protein in adult mouse RTN neurons using antibodies against its ECD (7C11) and ICD (SC-348). F, Overlay shows numerous NRG puncta (colabeled with 7C11 and SC-348) on or near cell bodies of PV-positive GABAergic neurons. Boxed ROI is magnified in G–J to illustrate the extensive overlap between ECD and ICD signals (arrowheads). DAPI is included in all images to label nuclei. Scale bars: A, B, 200 μm; C–J, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Fluorescence, Expressing, Immunofluorescence

NRG1 Type II protein accumulates at spinal chord C-boutons. Triple immunofluorescence histochemistry of Type II NRG1 protein in adult mouse spinal chord α-motorneuron cell bodies using antibodies against Type II NRG1 ECD (7C11) and ICD (SC-348), as well as the vAChT to identify cholinergic C-boutons. Arrowheads indicate examples of Type II NRG1 puncta at C-boutons labeled with both 7C11 and SC-348 that likely represent unprocessed pro-NRG1 at SSCs. Scale bar, 20 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: NRG1 Type II protein accumulates at spinal chord C-boutons. Triple immunofluorescence histochemistry of Type II NRG1 protein in adult mouse spinal chord α-motorneuron cell bodies using antibodies against Type II NRG1 ECD (7C11) and ICD (SC-348), as well as the vAChT to identify cholinergic C-boutons. Arrowheads indicate examples of Type II NRG1 puncta at C-boutons labeled with both 7C11 and SC-348 that likely represent unprocessed pro-NRG1 at SSCs. Scale bar, 20 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Immunofluorescence, Labeling

NRG3 is a dual-pass transmembrane domain protein. A, The presence of an additional transmembrane domain (TMN) in mouse NRG3 (accession number: NP_032760.1) between residues 69 and 91 is predicted by a hidden Markov model (TMHMM2.0 program) (Krogh et al., 2001). As in CRD-NRG1, sequences upstream of TMN and downstream of TMC are therefore predicted to be intracellular, whereas sequences between TMN and TMC, including the EGF-like domain, are extracellular. B, Analysis of NRG3 membrane topology in transfected HEK293 cells. Left, The NRG3 construct was V5-tagged near the EGF-like domain and HA-tagged immediately upstream of the TMN. Right, Under permeabilizing conditions, both anti-V5 and anti-HA antibodies bind NRG3/HA-V5. Under nonpermeabilizing conditions (i.e., surface labeling), only V5 immunoreactivity is observed. C, HEK293 cells transfected with NRG3-NTD-GFP, comprised of NRG3 sequences between the N terminus and the TMN fused to GFP (left), are labeled with anti-GFP under both nonpermeabilizing (Surface) and permeabilizing conditions, consistent with the notion that the N terminus harbors a transmembrane domain that tethers GFP to the plasma membrane. All NRG3 constructs were in pDESTDV3. B, C, Confocal images are from a single plane. Scale bars, 10 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: NRG3 is a dual-pass transmembrane domain protein. A, The presence of an additional transmembrane domain (TMN) in mouse NRG3 (accession number: NP_032760.1) between residues 69 and 91 is predicted by a hidden Markov model (TMHMM2.0 program) (Krogh et al., 2001). As in CRD-NRG1, sequences upstream of TMN and downstream of TMC are therefore predicted to be intracellular, whereas sequences between TMN and TMC, including the EGF-like domain, are extracellular. B, Analysis of NRG3 membrane topology in transfected HEK293 cells. Left, The NRG3 construct was V5-tagged near the EGF-like domain and HA-tagged immediately upstream of the TMN. Right, Under permeabilizing conditions, both anti-V5 and anti-HA antibodies bind NRG3/HA-V5. Under nonpermeabilizing conditions (i.e., surface labeling), only V5 immunoreactivity is observed. C, HEK293 cells transfected with NRG3-NTD-GFP, comprised of NRG3 sequences between the N terminus and the TMN fused to GFP (left), are labeled with anti-GFP under both nonpermeabilizing (Surface) and permeabilizing conditions, consistent with the notion that the N terminus harbors a transmembrane domain that tethers GFP to the plasma membrane. All NRG3 constructs were in pDESTDV3. B, C, Confocal images are from a single plane. Scale bars, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Transfection, Construct, Labeling

Axonal NRG3/CRD-NRG1 puncta are sites of juxtracrine interactions with ErbB4. A, Hippocampal neurons were transfected with NGR3/V5 and GFP as detailed in Figure 8 and labeled with anti-V5 and anti-ErbB4 antibodies. The representative low-magnification image shows a GFP-positive and ErbB4-negative transfected neuron next to an ErbB4-positive untransfected neuron. The two magnified ROIs illustrate how the transfected neuron lacks V5 immunoreactivity on its cell body (ROI 1) but harbors numerous V5 puncta on processes that overlap with ErbB4 signals on the untransfected neuron (ROI 1 and ROI 2, arrowheads). B, Quantitative analysis of colocalization of V5-tagged NRG3 and CRD-NRG1 with endogenous ErbB4 in transfected hippocampal neurons at DIV 10 and DIV 21. Data are mean ± SEM of 10 ROIs each from three separate experiments. C, V5 puncta reflecting accumulation of NRG3 (left) or CRD-NRG1 (right) on GFP-positive processes from transfected DIV 10 neurons (top) are absent from cultures treated with the NRG1 EGF-like domain (bottom). D, Top, Schematic illustration of the chimeric TGF_NRG3/V5 construct. Bottom, Anti-V5 Western blots confirming comparable expression of wild-type NRG3/V5 (WT) and TGF_NRG3/V5 (TGF) in transfected HEK 293 cells and in AAV-transduced cultured neurons. Blots were reprobed with anti-GAPDH to confirm equal loading. Untransfected/untransduced controls (Ctrl) were included to confirm specificity of the NRG3 bands. E, Axonal process from a neuron cotransfected with TGF_NRG3/V5 and CRD-NRG1/HA (included as internal reference). Unlike normal NRG3, which colocalizes with CRD-NRG1 (Fig. 8C), TGF_NRG3/V5 is absent from axonal varicosities that harbor CRD-NRG1/HA puncta. Scale bars: A, Overview image, 20 μm; A, Magnified ROIs, 10 μm; C, E, 5 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Axonal NRG3/CRD-NRG1 puncta are sites of juxtracrine interactions with ErbB4. A, Hippocampal neurons were transfected with NGR3/V5 and GFP as detailed in Figure 8 and labeled with anti-V5 and anti-ErbB4 antibodies. The representative low-magnification image shows a GFP-positive and ErbB4-negative transfected neuron next to an ErbB4-positive untransfected neuron. The two magnified ROIs illustrate how the transfected neuron lacks V5 immunoreactivity on its cell body (ROI 1) but harbors numerous V5 puncta on processes that overlap with ErbB4 signals on the untransfected neuron (ROI 1 and ROI 2, arrowheads). B, Quantitative analysis of colocalization of V5-tagged NRG3 and CRD-NRG1 with endogenous ErbB4 in transfected hippocampal neurons at DIV 10 and DIV 21. Data are mean ± SEM of 10 ROIs each from three separate experiments. C, V5 puncta reflecting accumulation of NRG3 (left) or CRD-NRG1 (right) on GFP-positive processes from transfected DIV 10 neurons (top) are absent from cultures treated with the NRG1 EGF-like domain (bottom). D, Top, Schematic illustration of the chimeric TGF_NRG3/V5 construct. Bottom, Anti-V5 Western blots confirming comparable expression of wild-type NRG3/V5 (WT) and TGF_NRG3/V5 (TGF) in transfected HEK 293 cells and in AAV-transduced cultured neurons. Blots were reprobed with anti-GAPDH to confirm equal loading. Untransfected/untransduced controls (Ctrl) were included to confirm specificity of the NRG3 bands. E, Axonal process from a neuron cotransfected with TGF_NRG3/V5 and CRD-NRG1/HA (included as internal reference). Unlike normal NRG3, which colocalizes with CRD-NRG1 (Fig. 8C), TGF_NRG3/V5 is absent from axonal varicosities that harbor CRD-NRG1/HA puncta. Scale bars: A, Overview image, 20 μm; A, Magnified ROIs, 10 μm; C, E, 5 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Transfection, Labeling, Construct, Western Blot, Expressing, Cell Culture

BACE inhibition prevents axonal accumulation of NRG3 and CRD-NRG1. A, Hippocampal neurons were cotransfected with GFP and NRG3/V5 or CRD-NRG1 as described in Figure 8, and labeled with anti-ECD (V5) and antibodies 1352 (NRG3) or SC-348 (NRG1) against their respective carboxyl-terminal ICDs. Representative images show processes of transfected (GFP-positive) neurons harboring punctate V5 signals (top) but lacking corresponding ICD signals (bottom), indicative of the accumulation of the processed rather than the unprocessed proteins. B–D, Anti-V5 Western blots of whole-cell lysates from neurons transduced with AAVs harboring NRG3/V5 or CRD-NRG1. B, Processed proteins (arrows) migrate at ∼70 kDa (NRG3) and ∼50 kDa (CRD-NRG1) apparent molecular mass, whereas their corresponding unprocessed proforms (barely detectable for NRG3) migrate at ∼130–140 kDa apparent molecular mass (arrowhead). C, Chronic (4 day) BACE inhibition with BACE-IV (1 μm), but not of matrix metalloproteinases with GM6001 (10 μm), blocks processing of NRG3 and CRD-NRG1 in transduced neurons. D, NMDA receptor blockade by AP5 (100 μm for 24 h) does not inhibit processing of dual-pass NRGs. E–H, BACE inhibition blocks axonal accumulation of NRG3 and CRD-NRG1 and causes retention of their unprocessed proforms in the cell body. Hippocampal neurons were cotransfected with GFP and NRG3/V5 (E, F) or with GFP and CRD-NRG1/V5 (G, H), and treated for 4 d with 1 μm BACE-IV. Untreated neurons were included as controls (Con). Neurons were labeled with antibodies against the ECD (V5) and ErbB4 to analyze NRG accumulation on axonal processes (E, G), or with anti-V5 and antibodies against the respective carboxyl-terminal domains of NRG3 (F) or NRG1 (H) to analyze cell body accumulation observed in a subset of transfected neurons (F, H). Scale bars: A, E, G, 5 μm; F, H, 10 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: BACE inhibition prevents axonal accumulation of NRG3 and CRD-NRG1. A, Hippocampal neurons were cotransfected with GFP and NRG3/V5 or CRD-NRG1 as described in Figure 8, and labeled with anti-ECD (V5) and antibodies 1352 (NRG3) or SC-348 (NRG1) against their respective carboxyl-terminal ICDs. Representative images show processes of transfected (GFP-positive) neurons harboring punctate V5 signals (top) but lacking corresponding ICD signals (bottom), indicative of the accumulation of the processed rather than the unprocessed proteins. B–D, Anti-V5 Western blots of whole-cell lysates from neurons transduced with AAVs harboring NRG3/V5 or CRD-NRG1. B, Processed proteins (arrows) migrate at ∼70 kDa (NRG3) and ∼50 kDa (CRD-NRG1) apparent molecular mass, whereas their corresponding unprocessed proforms (barely detectable for NRG3) migrate at ∼130–140 kDa apparent molecular mass (arrowhead). C, Chronic (4 day) BACE inhibition with BACE-IV (1 μm), but not of matrix metalloproteinases with GM6001 (10 μm), blocks processing of NRG3 and CRD-NRG1 in transduced neurons. D, NMDA receptor blockade by AP5 (100 μm for 24 h) does not inhibit processing of dual-pass NRGs. E–H, BACE inhibition blocks axonal accumulation of NRG3 and CRD-NRG1 and causes retention of their unprocessed proforms in the cell body. Hippocampal neurons were cotransfected with GFP and NRG3/V5 (E, F) or with GFP and CRD-NRG1/V5 (G, H), and treated for 4 d with 1 μm BACE-IV. Untreated neurons were included as controls (Con). Neurons were labeled with antibodies against the ECD (V5) and ErbB4 to analyze NRG accumulation on axonal processes (E, G), or with anti-V5 and antibodies against the respective carboxyl-terminal domains of NRG3 (F) or NRG1 (H) to analyze cell body accumulation observed in a subset of transfected neurons (F, H). Scale bars: A, E, G, 5 μm; F, H, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Inhibition, Labeling, Transfection, Western Blot, Transduction

Axonal accumulation is impaired in cleavage-resistant variants of NRG3 and CRD-NRG1. A, Schematic illustration of the strategy to render dual-pass NRGs resistant to processing near the TMC. Briefly, the area between the EGF-like and TM domains of NRG3 and CRD-NRG1 was replaced by the shedding-resistant juxtamembrane region of the JM-b isoform of ErbB4 (Elenius et al., 1997). B, Western blot analysis of whole-cell lysates from hippocampal neurons transduced with AAVs harboring cleavable (wt) or cleavage-resistant (cr) variants of CRD-NRG1/V5 and NRG3/V5. Arrowhead indicates bands representing uncleaved proforms. Arrows indicate processed proteins. C, D, Lack of axonal accumulation of cleavage-resistant NRG variants in neurons transfected with crNRG3/V5 (C) or crCRD1-NRG1 (D) along with GFP and wtCRD-NRG1/HA (included as internal reference). Top, Neurons were triple-labeled with anti-ErbB4, anti-HA to reveal sites of axonal CRD-NRG1 accumulation, and anti-V5 to detect crNRG variants. GFP-positive axonal processes forming varicosities across from ErbB4 puncta (left) harbor wtCRD-NRG1 puncta (right) but lack corresponding signals for crNRG3 or crCRD-NRG1 (middle). Bottom, In parallel experiments, transfected neurons were labeled with antibodies against the ECDs (V5) and the carboxyl-terminal ICDs of crNRG3 or CRD-NRG1, as described in Figure 10. Representative images of transfected neuron cell bodies (left) show accumulation of crNRGs with both ECD (middle) and ICD (right) antibodies. E, Representative confocal image of a crNRG3/V5-expressing hippocampal neuron labeled with anti-V5 and an antibody against the trans-Golgi marker protein TGN-38. F, Neurons were transfected with GFP and V5-tagged NRG3(Q360*) lacking the TMC and downstream sequences. Representative confocal images of GFP-positive processes from transfected neurons labeled for V5 and ErbB4 showing NRG3(Q360*)-puncta under untreated control conditions (top) and after BACE-IV treatment (bottom). Scale bars: C, Top, D, Top, 5 μm; C, Bottom, D, Bottom, E, F, 10 μm.

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: Axonal accumulation is impaired in cleavage-resistant variants of NRG3 and CRD-NRG1. A, Schematic illustration of the strategy to render dual-pass NRGs resistant to processing near the TMC. Briefly, the area between the EGF-like and TM domains of NRG3 and CRD-NRG1 was replaced by the shedding-resistant juxtamembrane region of the JM-b isoform of ErbB4 (Elenius et al., 1997). B, Western blot analysis of whole-cell lysates from hippocampal neurons transduced with AAVs harboring cleavable (wt) or cleavage-resistant (cr) variants of CRD-NRG1/V5 and NRG3/V5. Arrowhead indicates bands representing uncleaved proforms. Arrows indicate processed proteins. C, D, Lack of axonal accumulation of cleavage-resistant NRG variants in neurons transfected with crNRG3/V5 (C) or crCRD1-NRG1 (D) along with GFP and wtCRD-NRG1/HA (included as internal reference). Top, Neurons were triple-labeled with anti-ErbB4, anti-HA to reveal sites of axonal CRD-NRG1 accumulation, and anti-V5 to detect crNRG variants. GFP-positive axonal processes forming varicosities across from ErbB4 puncta (left) harbor wtCRD-NRG1 puncta (right) but lack corresponding signals for crNRG3 or crCRD-NRG1 (middle). Bottom, In parallel experiments, transfected neurons were labeled with antibodies against the ECDs (V5) and the carboxyl-terminal ICDs of crNRG3 or CRD-NRG1, as described in Figure 10. Representative images of transfected neuron cell bodies (left) show accumulation of crNRGs with both ECD (middle) and ICD (right) antibodies. E, Representative confocal image of a crNRG3/V5-expressing hippocampal neuron labeled with anti-V5 and an antibody against the trans-Golgi marker protein TGN-38. F, Neurons were transfected with GFP and V5-tagged NRG3(Q360*) lacking the TMC and downstream sequences. Representative confocal images of GFP-positive processes from transfected neurons labeled for V5 and ErbB4 showing NRG3(Q360*)-puncta under untreated control conditions (top) and after BACE-IV treatment (bottom). Scale bars: C, Top, D, Top, 5 μm; C, Bottom, D, Bottom, E, F, 10 μm.

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Western Blot, Transduction, Transfection, Labeling, Expressing, Marker

The relationship between transmembrane topology and protein processing in the differential accumulation of NRG isoforms on the cell body versus axons of central neurons. This model is largely based on data obtained in cultured hippocampal neurons expressing low to moderate NRG levels; more widespread distribution is possible in neurons expressing higher levels. A, Single-pass NRGs accumulate at SSC-type ER-PM contact sites on cell bodies and proximal dendrites and act as local paracrine or autocrine signals upon ectodomain shedding. By contrast, processed dual-pass NRGs accumulate on axons and axon terminals where they act as distal signals to modulate synaptic function or to regulate neuron–glia interactions via juxtacrine interactions with ErbB receptors. Importantly, local and distal NRG signaling might occur simultaneously in neurons coexpressing both single- and dual-pass NRGs. B, Single-pass NRGs are trafficked through the secretory pathway as unprocessed proforms and accumulate at SSC-type ER-PM contact sites. Ectodomain shedding in response to glutamate signaling via NMDA receptors occurs at the neuronal cell surface and is mediated by GM6001-sensitive metalloproteinases, likely ADAM10 or ADAM17/TACE (Montero et al., 2000; Horiuchi et al., 2005; Fleck et al., 2013). Interactions between the Ig-like domain and the extracellular matrix limit the spread of the ectodomain and thereby promote locally restricted signaling. By contrast, processing of dual-pass NRGs by BACE, likely in the trans-Golgi network (Fig. 11E) or in Golgi-associated vesicles, generates an amino-terminal fragment that accumulates on axons via juxtracrine ErbB interactions. Furthermore, both single- and dual-pass NRGs give rise to intracellular carboxyl-terminal proteolytic fragments that result from additional processing by γ-secretase. For CRD-NRG1, back signaling by this fragment has been reported to acutely regulate TRPV and α7-containing nAChRs in axons (Hancock et al., 2008; Canetta et al., 2011) as well as gene expression in the nucleus (Bao et al., 2003, 2004).

Journal: The Journal of Neuroscience

Article Title: Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

doi: 10.1523/JNEUROSCI.2630-16.2017

Figure Lengend Snippet: The relationship between transmembrane topology and protein processing in the differential accumulation of NRG isoforms on the cell body versus axons of central neurons. This model is largely based on data obtained in cultured hippocampal neurons expressing low to moderate NRG levels; more widespread distribution is possible in neurons expressing higher levels. A, Single-pass NRGs accumulate at SSC-type ER-PM contact sites on cell bodies and proximal dendrites and act as local paracrine or autocrine signals upon ectodomain shedding. By contrast, processed dual-pass NRGs accumulate on axons and axon terminals where they act as distal signals to modulate synaptic function or to regulate neuron–glia interactions via juxtacrine interactions with ErbB receptors. Importantly, local and distal NRG signaling might occur simultaneously in neurons coexpressing both single- and dual-pass NRGs. B, Single-pass NRGs are trafficked through the secretory pathway as unprocessed proforms and accumulate at SSC-type ER-PM contact sites. Ectodomain shedding in response to glutamate signaling via NMDA receptors occurs at the neuronal cell surface and is mediated by GM6001-sensitive metalloproteinases, likely ADAM10 or ADAM17/TACE (Montero et al., 2000; Horiuchi et al., 2005; Fleck et al., 2013). Interactions between the Ig-like domain and the extracellular matrix limit the spread of the ectodomain and thereby promote locally restricted signaling. By contrast, processing of dual-pass NRGs by BACE, likely in the trans-Golgi network (Fig. 11E) or in Golgi-associated vesicles, generates an amino-terminal fragment that accumulates on axons via juxtracrine ErbB interactions. Furthermore, both single- and dual-pass NRGs give rise to intracellular carboxyl-terminal proteolytic fragments that result from additional processing by γ-secretase. For CRD-NRG1, back signaling by this fragment has been reported to acutely regulate TRPV and α7-containing nAChRs in axons (Hancock et al., 2008; Canetta et al., 2011) as well as gene expression in the nucleus (Bao et al., 2003, 2004).

Article Snippet: Recombinant Type II NRG1-ECD was then used to immunize SJL mice and to develop mouse monoclonal anti-NRG1 antibody 7C11 (hybridomas generated at Precision Antibodies).

Techniques: Cell Culture, Expressing

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