neurotrophin 3  (Alomone Labs)


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    Structured Review

    Alomone Labs neurotrophin 3
    The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of <t>neurotrophin</t> 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.
    Neurotrophin 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    neurotrophin 3 - by Bioz Stars, 2022-12
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    Images

    1) Product Images from "Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats"

    Article Title: Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104759

    The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of neurotrophin 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.
    Figure Legend Snippet: The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of neurotrophin 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.

    Techniques Used: Expressing, Western Blot, Derivative Assay

    2) Product Images from "Involvement of NT3 and P75NTR in photoreceptor degeneration following selective M?ller cell ablation"

    Article Title: Involvement of NT3 and P75NTR in photoreceptor degeneration following selective M?ller cell ablation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-10-137

    Inhibition of microglial activation after intravitreal injection of recombinant mature NT3 and blockage of P75 NTR . Transgenic mice received twice intravitreal injections of NT3 (0.4 μg), P75 NTR rabbit polyclonal a polyclonal Ab to P75 NTR (1:1 dilution), a combination of both or balanced salt solution ( BSS ) 3d and 6d after tamoxifen (TMX)-induced Muller cell ablation. Eyes were enucleated 10d after TMX treatment for retinal flat-mount staining using an Ab against ionized calcium binding adaptor molecule 1 ( IBA-1 ) for microglia ( A, D, G and J , green ) and peanut-agglutinin ( PNA )-conjugated with Alexa Fluor-594 for cone photoreceptor outer segments ( B, E, H and K , red ). (C, F, I and L) Merged images. (M) Quantitative analysis of IBA-1 stained retinal whole mounts shows intravitreal injection of NT3 and P75 NTR blockage, either alone or in combination, protected photoreceptors concurrently with inhibition of microglial activation. * P
    Figure Legend Snippet: Inhibition of microglial activation after intravitreal injection of recombinant mature NT3 and blockage of P75 NTR . Transgenic mice received twice intravitreal injections of NT3 (0.4 μg), P75 NTR rabbit polyclonal a polyclonal Ab to P75 NTR (1:1 dilution), a combination of both or balanced salt solution ( BSS ) 3d and 6d after tamoxifen (TMX)-induced Muller cell ablation. Eyes were enucleated 10d after TMX treatment for retinal flat-mount staining using an Ab against ionized calcium binding adaptor molecule 1 ( IBA-1 ) for microglia ( A, D, G and J , green ) and peanut-agglutinin ( PNA )-conjugated with Alexa Fluor-594 for cone photoreceptor outer segments ( B, E, H and K , red ). (C, F, I and L) Merged images. (M) Quantitative analysis of IBA-1 stained retinal whole mounts shows intravitreal injection of NT3 and P75 NTR blockage, either alone or in combination, protected photoreceptors concurrently with inhibition of microglial activation. * P

    Techniques Used: Inhibition, Activation Assay, Injection, Recombinant, Transgenic Assay, Mouse Assay, Staining, Binding Assay

    Differential expression of mature NT3, pro-NT3, rod and cone phototransduction proteins 7d after Müller cell ablation. (A, F) Western blots using antibodies to detect mature NT3, pro-NT3, rhodopsin, guanine nucleotide-binding protein subunit alpha-1 (GNAT1) and Gα protein transducin (Gαt). GNAT1 and Gαt are proteins essential for rod and cone phototransduction [ 27 - 29 ]. (B-D) Quantitative analysis of protein densitometry showed significant reduction in mature NT3 (B) and upregulation of pro-NT3 (C) , which resulted in a decreased ratio of NT3:pro-NT3 (D) 7d after Müller cell ablation. (E, G, H) The reduced expression of rhodopsin (E) , GNAT1 (G) and Gαt (H) in transgenic (TG) mice indicates that selective Müller cell ablation causes damage to both rod and cone photoreceptors. * P
    Figure Legend Snippet: Differential expression of mature NT3, pro-NT3, rod and cone phototransduction proteins 7d after Müller cell ablation. (A, F) Western blots using antibodies to detect mature NT3, pro-NT3, rhodopsin, guanine nucleotide-binding protein subunit alpha-1 (GNAT1) and Gα protein transducin (Gαt). GNAT1 and Gαt are proteins essential for rod and cone phototransduction [ 27 - 29 ]. (B-D) Quantitative analysis of protein densitometry showed significant reduction in mature NT3 (B) and upregulation of pro-NT3 (C) , which resulted in a decreased ratio of NT3:pro-NT3 (D) 7d after Müller cell ablation. (E, G, H) The reduced expression of rhodopsin (E) , GNAT1 (G) and Gαt (H) in transgenic (TG) mice indicates that selective Müller cell ablation causes damage to both rod and cone photoreceptors. * P

    Techniques Used: Expressing, Western Blot, Binding Assay, Transgenic Assay, Mouse Assay

    Intravitreal injection of recombinant NT3 and blockage of P75 NTR protected photoreceptor degeneration. Intravitreal injection was performed in transgenic mice 3d and 6d after tamoxifen induced Müller cell ablation, with one eye receiving the testing reagent and the contralateral eye receiving balanced salt solution (BSS). Doses of injection: (1) NT3, 0.4 μg; (2) P75 NTR rabbit polyclonal Ab (1:1 dilution) and (3) 0.4 μg NT3 + 1:1 dilution of P75 NTR Ab. Eyes were enucleated 10d after TMX treatment for flat-mount staining using fluorescence-conjugated peanut-agglutinin (PNA). (A and B) PNA-labeled cone photoreceptor outer segments ( green ) and nuclear counterstaining in a control ( Ctl ) retina. (C-F) Images from transgenic ( TG ) mice showing changes in cone photoreceptor outer segments and protrusion of photoreceptor nuclei into the subretinal space ( red , indicated by asterisks ) in eyes receiving BSS (C) , NT3 (D) , P75 NTR Ab (E) and a combination of NT3 and P75 NTR Ab (F) . (G) Quantitative analysis of PNA staining showed that intravitreal injection of NT3 and P75 Ab protected the loss of cone photoreceptor outer segments, with a combined treatment more effective than either alone. N = 9 − 11/group. Scale bars in A-F : 100 μm.
    Figure Legend Snippet: Intravitreal injection of recombinant NT3 and blockage of P75 NTR protected photoreceptor degeneration. Intravitreal injection was performed in transgenic mice 3d and 6d after tamoxifen induced Müller cell ablation, with one eye receiving the testing reagent and the contralateral eye receiving balanced salt solution (BSS). Doses of injection: (1) NT3, 0.4 μg; (2) P75 NTR rabbit polyclonal Ab (1:1 dilution) and (3) 0.4 μg NT3 + 1:1 dilution of P75 NTR Ab. Eyes were enucleated 10d after TMX treatment for flat-mount staining using fluorescence-conjugated peanut-agglutinin (PNA). (A and B) PNA-labeled cone photoreceptor outer segments ( green ) and nuclear counterstaining in a control ( Ctl ) retina. (C-F) Images from transgenic ( TG ) mice showing changes in cone photoreceptor outer segments and protrusion of photoreceptor nuclei into the subretinal space ( red , indicated by asterisks ) in eyes receiving BSS (C) , NT3 (D) , P75 NTR Ab (E) and a combination of NT3 and P75 NTR Ab (F) . (G) Quantitative analysis of PNA staining showed that intravitreal injection of NT3 and P75 Ab protected the loss of cone photoreceptor outer segments, with a combined treatment more effective than either alone. N = 9 − 11/group. Scale bars in A-F : 100 μm.

    Techniques Used: Injection, Recombinant, Transgenic Assay, Mouse Assay, Staining, Fluorescence, Labeling, CTL Assay

    3) Product Images from "Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons"

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    Journal: The European journal of neuroscience

    doi: 10.1111/j.1460-9568.2010.07556.x

    Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.
    Figure Legend Snippet: Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Techniques Used: Binding Assay, Transfection, Incubation, Immunofluorescence

    Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3
    Figure Legend Snippet: Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Techniques Used: Transfection, Variant Assay, Incubation, Immunoprecipitation, SDS Page, Expressing

    Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5
    Figure Legend Snippet: Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Techniques Used: SDS Page, Protein Concentration, Recombinant

    ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.
    Figure Legend Snippet: ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Techniques Used: Incubation

    SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance
    Figure Legend Snippet: SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Techniques Used: SPR Assay, Ligand Binding Assay, Sequencing

    4) Product Images from "Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons"

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    Journal: The European journal of neuroscience

    doi: 10.1111/j.1460-9568.2010.07556.x

    ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.
    Figure Legend Snippet: ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.

    Techniques Used: Cell Culture, Incubation, Modification, Staining, Marker, Standard Deviation

    Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.
    Figure Legend Snippet: Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Techniques Used: Binding Assay, Transfection, Incubation, Immunofluorescence

    Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3
    Figure Legend Snippet: Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Techniques Used: Transfection, Variant Assay, Incubation, Immunoprecipitation, SDS Page, Expressing

    Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5
    Figure Legend Snippet: Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Techniques Used: SDS Page, Protein Concentration, Recombinant

    ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.
    Figure Legend Snippet: ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Techniques Used: Incubation

    SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance
    Figure Legend Snippet: SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Techniques Used: SPR Assay, Ligand Binding Assay, Sequencing

    5) Product Images from "Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons"

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    Journal: The European journal of neuroscience

    doi: 10.1111/j.1460-9568.2010.07556.x

    ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.
    Figure Legend Snippet: ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.

    Techniques Used: Cell Culture, Incubation, Modification, Staining, Marker, Standard Deviation

    Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.
    Figure Legend Snippet: Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Techniques Used: Binding Assay, Transfection, Incubation, Immunofluorescence

    Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3
    Figure Legend Snippet: Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Techniques Used: Transfection, Variant Assay, Incubation, Immunoprecipitation, SDS Page, Expressing

    Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5
    Figure Legend Snippet: Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Techniques Used: SDS Page, Protein Concentration, Recombinant

    ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.
    Figure Legend Snippet: ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Techniques Used: Incubation

    SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance
    Figure Legend Snippet: SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Techniques Used: SPR Assay, Ligand Binding Assay, Sequencing

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    Alomone Labs neurotrophin 3
    The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of <t>neurotrophin</t> 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.
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    The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of neurotrophin 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.

    Journal: PLoS ONE

    Article Title: Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats

    doi: 10.1371/journal.pone.0104759

    Figure Lengend Snippet: The effects of EPO on differential expression of p75 NTR , pro-NT3 and VEGF-A. (A and C) Western blots using antibodies to detect p75 neutrotropin receptor (p75 NTR ), the precursor form of neurotrophin 3 (pro-NT3), tumor necrosis factor alpha (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). (B, D) Quantitative analysis of protein band densitometry, n = 4/group.

    Article Snippet: Membranes were probed with primary antibodies to GFAP (goat polyclonal, 1∶500, Abcam, #ab53554), glutamine synthetase (GS, mouse monoclonal, 1∶1000, Millipore #MAB302), p75 neurotrophin receptor (p75NTR , rabbit polyclonal, 1;1000, Covance #PRB-602C), precursor form of neurotrophin 3 (pro-NT3, rabbit polyclonal, 1∶500, Alomone laboratory #ANT-012), tumour necrosis factor-α (TNFα, rabbit polyclonal, 1∶2000, Millipore #AB2148P), pigment epithelium derived factor (PEDF, goat polyclonal, R & D Systems # AF1149) and vascular endothelial growth factor –A (VEGF-A, mouse monoclonal, 1∶1000, Abcam #ab1316).

    Techniques: Expressing, Western Blot, Derivative Assay

    Inhibition of microglial activation after intravitreal injection of recombinant mature NT3 and blockage of P75 NTR . Transgenic mice received twice intravitreal injections of NT3 (0.4 μg), P75 NTR rabbit polyclonal a polyclonal Ab to P75 NTR (1:1 dilution), a combination of both or balanced salt solution ( BSS ) 3d and 6d after tamoxifen (TMX)-induced Muller cell ablation. Eyes were enucleated 10d after TMX treatment for retinal flat-mount staining using an Ab against ionized calcium binding adaptor molecule 1 ( IBA-1 ) for microglia ( A, D, G and J , green ) and peanut-agglutinin ( PNA )-conjugated with Alexa Fluor-594 for cone photoreceptor outer segments ( B, E, H and K , red ). (C, F, I and L) Merged images. (M) Quantitative analysis of IBA-1 stained retinal whole mounts shows intravitreal injection of NT3 and P75 NTR blockage, either alone or in combination, protected photoreceptors concurrently with inhibition of microglial activation. * P

    Journal: Journal of Neuroinflammation

    Article Title: Involvement of NT3 and P75NTR in photoreceptor degeneration following selective M?ller cell ablation

    doi: 10.1186/1742-2094-10-137

    Figure Lengend Snippet: Inhibition of microglial activation after intravitreal injection of recombinant mature NT3 and blockage of P75 NTR . Transgenic mice received twice intravitreal injections of NT3 (0.4 μg), P75 NTR rabbit polyclonal a polyclonal Ab to P75 NTR (1:1 dilution), a combination of both or balanced salt solution ( BSS ) 3d and 6d after tamoxifen (TMX)-induced Muller cell ablation. Eyes were enucleated 10d after TMX treatment for retinal flat-mount staining using an Ab against ionized calcium binding adaptor molecule 1 ( IBA-1 ) for microglia ( A, D, G and J , green ) and peanut-agglutinin ( PNA )-conjugated with Alexa Fluor-594 for cone photoreceptor outer segments ( B, E, H and K , red ). (C, F, I and L) Merged images. (M) Quantitative analysis of IBA-1 stained retinal whole mounts shows intravitreal injection of NT3 and P75 NTR blockage, either alone or in combination, protected photoreceptors concurrently with inhibition of microglial activation. * P

    Article Snippet: Membranes were probed with antibodies to GS (mouse monoclonal, 1:1,000; Millipore, no. MAB302), GFAP (mouse monoclonal, 1:5,000; Neomarker, no. MS-280-P), P75NTR (rabbit polyclonal, a gift from Dr. Moses V. Chao, no. 9651), NT3 (rabbit polyclonal, 1:1,000, Alomone Laboratory, no. ANT-003), pro-NT3 (rabbit polyclonal, 1:500, Alomone Laboratory, no. ANT-012) and rhodopsin (mouse monoclonal, 1:500, Millipore no. MAB5356), guanine nucleotide-binding protein subunit alpha-1 (GNAT1, rabbit polyclonal, 1:500, Santa Cruz no. sc-389) and Gα protein transducin (Gαt, mouse monoclonal, 1:2,000, BD Transduction Laboratories no. 610589).

    Techniques: Inhibition, Activation Assay, Injection, Recombinant, Transgenic Assay, Mouse Assay, Staining, Binding Assay

    Differential expression of mature NT3, pro-NT3, rod and cone phototransduction proteins 7d after Müller cell ablation. (A, F) Western blots using antibodies to detect mature NT3, pro-NT3, rhodopsin, guanine nucleotide-binding protein subunit alpha-1 (GNAT1) and Gα protein transducin (Gαt). GNAT1 and Gαt are proteins essential for rod and cone phototransduction [ 27 - 29 ]. (B-D) Quantitative analysis of protein densitometry showed significant reduction in mature NT3 (B) and upregulation of pro-NT3 (C) , which resulted in a decreased ratio of NT3:pro-NT3 (D) 7d after Müller cell ablation. (E, G, H) The reduced expression of rhodopsin (E) , GNAT1 (G) and Gαt (H) in transgenic (TG) mice indicates that selective Müller cell ablation causes damage to both rod and cone photoreceptors. * P

    Journal: Journal of Neuroinflammation

    Article Title: Involvement of NT3 and P75NTR in photoreceptor degeneration following selective M?ller cell ablation

    doi: 10.1186/1742-2094-10-137

    Figure Lengend Snippet: Differential expression of mature NT3, pro-NT3, rod and cone phototransduction proteins 7d after Müller cell ablation. (A, F) Western blots using antibodies to detect mature NT3, pro-NT3, rhodopsin, guanine nucleotide-binding protein subunit alpha-1 (GNAT1) and Gα protein transducin (Gαt). GNAT1 and Gαt are proteins essential for rod and cone phototransduction [ 27 - 29 ]. (B-D) Quantitative analysis of protein densitometry showed significant reduction in mature NT3 (B) and upregulation of pro-NT3 (C) , which resulted in a decreased ratio of NT3:pro-NT3 (D) 7d after Müller cell ablation. (E, G, H) The reduced expression of rhodopsin (E) , GNAT1 (G) and Gαt (H) in transgenic (TG) mice indicates that selective Müller cell ablation causes damage to both rod and cone photoreceptors. * P

    Article Snippet: Membranes were probed with antibodies to GS (mouse monoclonal, 1:1,000; Millipore, no. MAB302), GFAP (mouse monoclonal, 1:5,000; Neomarker, no. MS-280-P), P75NTR (rabbit polyclonal, a gift from Dr. Moses V. Chao, no. 9651), NT3 (rabbit polyclonal, 1:1,000, Alomone Laboratory, no. ANT-003), pro-NT3 (rabbit polyclonal, 1:500, Alomone Laboratory, no. ANT-012) and rhodopsin (mouse monoclonal, 1:500, Millipore no. MAB5356), guanine nucleotide-binding protein subunit alpha-1 (GNAT1, rabbit polyclonal, 1:500, Santa Cruz no. sc-389) and Gα protein transducin (Gαt, mouse monoclonal, 1:2,000, BD Transduction Laboratories no. 610589).

    Techniques: Expressing, Western Blot, Binding Assay, Transgenic Assay, Mouse Assay

    Intravitreal injection of recombinant NT3 and blockage of P75 NTR protected photoreceptor degeneration. Intravitreal injection was performed in transgenic mice 3d and 6d after tamoxifen induced Müller cell ablation, with one eye receiving the testing reagent and the contralateral eye receiving balanced salt solution (BSS). Doses of injection: (1) NT3, 0.4 μg; (2) P75 NTR rabbit polyclonal Ab (1:1 dilution) and (3) 0.4 μg NT3 + 1:1 dilution of P75 NTR Ab. Eyes were enucleated 10d after TMX treatment for flat-mount staining using fluorescence-conjugated peanut-agglutinin (PNA). (A and B) PNA-labeled cone photoreceptor outer segments ( green ) and nuclear counterstaining in a control ( Ctl ) retina. (C-F) Images from transgenic ( TG ) mice showing changes in cone photoreceptor outer segments and protrusion of photoreceptor nuclei into the subretinal space ( red , indicated by asterisks ) in eyes receiving BSS (C) , NT3 (D) , P75 NTR Ab (E) and a combination of NT3 and P75 NTR Ab (F) . (G) Quantitative analysis of PNA staining showed that intravitreal injection of NT3 and P75 Ab protected the loss of cone photoreceptor outer segments, with a combined treatment more effective than either alone. N = 9 − 11/group. Scale bars in A-F : 100 μm.

    Journal: Journal of Neuroinflammation

    Article Title: Involvement of NT3 and P75NTR in photoreceptor degeneration following selective M?ller cell ablation

    doi: 10.1186/1742-2094-10-137

    Figure Lengend Snippet: Intravitreal injection of recombinant NT3 and blockage of P75 NTR protected photoreceptor degeneration. Intravitreal injection was performed in transgenic mice 3d and 6d after tamoxifen induced Müller cell ablation, with one eye receiving the testing reagent and the contralateral eye receiving balanced salt solution (BSS). Doses of injection: (1) NT3, 0.4 μg; (2) P75 NTR rabbit polyclonal Ab (1:1 dilution) and (3) 0.4 μg NT3 + 1:1 dilution of P75 NTR Ab. Eyes were enucleated 10d after TMX treatment for flat-mount staining using fluorescence-conjugated peanut-agglutinin (PNA). (A and B) PNA-labeled cone photoreceptor outer segments ( green ) and nuclear counterstaining in a control ( Ctl ) retina. (C-F) Images from transgenic ( TG ) mice showing changes in cone photoreceptor outer segments and protrusion of photoreceptor nuclei into the subretinal space ( red , indicated by asterisks ) in eyes receiving BSS (C) , NT3 (D) , P75 NTR Ab (E) and a combination of NT3 and P75 NTR Ab (F) . (G) Quantitative analysis of PNA staining showed that intravitreal injection of NT3 and P75 Ab protected the loss of cone photoreceptor outer segments, with a combined treatment more effective than either alone. N = 9 − 11/group. Scale bars in A-F : 100 μm.

    Article Snippet: Membranes were probed with antibodies to GS (mouse monoclonal, 1:1,000; Millipore, no. MAB302), GFAP (mouse monoclonal, 1:5,000; Neomarker, no. MS-280-P), P75NTR (rabbit polyclonal, a gift from Dr. Moses V. Chao, no. 9651), NT3 (rabbit polyclonal, 1:1,000, Alomone Laboratory, no. ANT-003), pro-NT3 (rabbit polyclonal, 1:500, Alomone Laboratory, no. ANT-012) and rhodopsin (mouse monoclonal, 1:500, Millipore no. MAB5356), guanine nucleotide-binding protein subunit alpha-1 (GNAT1, rabbit polyclonal, 1:500, Santa Cruz no. sc-389) and Gα protein transducin (Gαt, mouse monoclonal, 1:2,000, BD Transduction Laboratories no. 610589).

    Techniques: Injection, Recombinant, Transgenic Assay, Mouse Assay, Staining, Fluorescence, Labeling, CTL Assay

    Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Article Snippet: The blots were blocked in 5% defatted milk in TBST buffer (50mM Tris, 500mM NaCl, 0.1% Tween-20, pH 7.4) for 1 hour at RT and subsequently incubated over night at 4°C in blocking buffer with mouse anti-human Sortilin, rabbit anti-human p75NTR (provided by Moses V. Chao), rabbit anti-proNT3, rabbit anti-NT3 (Alomone Labs Ltd.) or mouse anti-beta-actin (Sigma Aldrich).

    Techniques: Binding Assay, Transfection, Incubation, Immunofluorescence

    Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Article Snippet: The blots were blocked in 5% defatted milk in TBST buffer (50mM Tris, 500mM NaCl, 0.1% Tween-20, pH 7.4) for 1 hour at RT and subsequently incubated over night at 4°C in blocking buffer with mouse anti-human Sortilin, rabbit anti-human p75NTR (provided by Moses V. Chao), rabbit anti-proNT3, rabbit anti-NT3 (Alomone Labs Ltd.) or mouse anti-beta-actin (Sigma Aldrich).

    Techniques: Transfection, Variant Assay, Incubation, Immunoprecipitation, SDS Page, Expressing

    Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Article Snippet: The blots were blocked in 5% defatted milk in TBST buffer (50mM Tris, 500mM NaCl, 0.1% Tween-20, pH 7.4) for 1 hour at RT and subsequently incubated over night at 4°C in blocking buffer with mouse anti-human Sortilin, rabbit anti-human p75NTR (provided by Moses V. Chao), rabbit anti-proNT3, rabbit anti-NT3 (Alomone Labs Ltd.) or mouse anti-beta-actin (Sigma Aldrich).

    Techniques: SDS Page, Protein Concentration, Recombinant

    ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Article Snippet: The blots were blocked in 5% defatted milk in TBST buffer (50mM Tris, 500mM NaCl, 0.1% Tween-20, pH 7.4) for 1 hour at RT and subsequently incubated over night at 4°C in blocking buffer with mouse anti-human Sortilin, rabbit anti-human p75NTR (provided by Moses V. Chao), rabbit anti-proNT3, rabbit anti-NT3 (Alomone Labs Ltd.) or mouse anti-beta-actin (Sigma Aldrich).

    Techniques: Incubation

    SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Article Snippet: The blots were blocked in 5% defatted milk in TBST buffer (50mM Tris, 500mM NaCl, 0.1% Tween-20, pH 7.4) for 1 hour at RT and subsequently incubated over night at 4°C in blocking buffer with mouse anti-human Sortilin, rabbit anti-human p75NTR (provided by Moses V. Chao), rabbit anti-proNT3, rabbit anti-NT3 (Alomone Labs Ltd.) or mouse anti-beta-actin (Sigma Aldrich).

    Techniques: SPR Assay, Ligand Binding Assay, Sequencing

    ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.

    Journal: The European journal of neuroscience

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    doi: 10.1111/j.1460-9568.2010.07556.x

    Figure Lengend Snippet: ProNT3 induced apoptosis in SG neurons ProNT3-induced apoptosis was also investigated in p75 NTR - and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.

    Article Snippet: Precipitated proteins and input lysates were analysed by reducing SDS-PAGE and western blotting using monoclonal anti-Sortilin (anti-Neurotensin R3 mAb, Transduction Laboratories), anti-proNT3 (Alomone Labs Ltd.), and anti-p75NTR .

    Techniques: Cell Culture, Incubation, Modification, Staining, Marker, Standard Deviation