prongf protein Search Results


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  • 99
    Thermo Fisher alexa fluor 594 labeling
    Alexa Fluor 594 Labeling, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sds polyacrylamide gel electrophoresis
    Sds Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad prongf peptides
    Histological localization of <t>NGF,</t> <t>proNGF</t> and their cognate receptors, TrkA and p75NTR, in parenchymal hepatocytes of human livers with hepatolithiasis. Liver sections of lithiasis and contralateral parts in hepatolithiasis liver tissues ( n =5) and paralesional sections of hepatocellular carcinoma livers ( n =4) as non-hepatolithiasis control (NHC) underwent formalin-fixation, tissue-processing and paraffin-embedding procedures. Tissue sections were subjected to immunohistochemical staining for NGF, proNGF, TrkA and p75NTR. Scale bars=100 μm. Arrows indicate characteristic fatty changes in lithiasis tissues, while white and black arrowheads indicate immunoreactive signals in the hepatocytes and the infiltrated cells, respectively.
    Prongf Peptides, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Scil Proteins wt prongf
    <t>WT-ProNGF</t> induces TrkA-dependent signaling cascades. PC12 cells ( panels A and B ) or PC12nnr5 cells ( panels C and D ) were incubated in DMEB for 1 h and then treated with 25 ng/ml <t>NGF</t> or 50 ng/ml WT-proNGF for the time points indicated. Cells were lysed
    Wt Prongf, supplied by Scil Proteins, used in various techniques. Bioz Stars score: 84/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore anti prongf
    SPR analysis—calibration curves obtained with <t>proNGF</t> and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb <t>Millipore.</t> Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.
    Anti Prongf, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Prospec recombinant prongf
    Expression and localization of p75 NTR and neurotoxic factors increase during the progression of DR. The experimental paradigm and the endpoints of the mouse DR model are shown. Drug treatments were injected intravitreally at week 2.5 of diabetes in this paradigm. A , p75 NTR , <t>proNGF,</t> <t>TNFα,</t> and α 2 M expression in whole mice retina analyzed by Western blot from 1–6 weeks after induction of diabetes. There were increases of p75 NTR during week 1, of proNGF during week 3, of TNFα during week 4, and of α 2 M during week 5. B , Quantification. * p
    Recombinant Prongf, supplied by Prospec, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA prongf
    Changes in adipose tissue expression of A) pro nerve growth factor <t>(proNGF),</t> B) mature NGF <t>(mNGF)</t> and C) the mNGF/proNGF ratio after 5 weeks of electroacupuncture (EA) measured by ELISA. D) Representative blot of each proNGF splicing variant from before and after 5 weeks of EA measured by western blot and corresponding densitometry analyses for E) proNGF 50 kDa; F) proNGF 34 kDa and G) all proNGF variants detectable in adipose tissue, and H) representative western blots and corresponding gel densitometry analyses for adipose tissue expression of the mNGF/proNGF receptors I) phosphorylated‐TrkA and J) p75 NTR .
    Prongf, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cedarlane recombinant prongf
    Stimulation of <t>GCs</t> with <t>proNGF</t> and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p
    Recombinant Prongf, supplied by Cedarlane, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Alomone Labs human recombinant wild type prongf
    Stimulation of <t>GCs</t> with <t>proNGF</t> and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p
    Human Recombinant Wild Type Prongf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Cell Sciences Inc recombinant human prongf treatment
    Stimulation of <t>GCs</t> with <t>proNGF</t> and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p
    Recombinant Human Prongf Treatment, supplied by Cell Sciences Inc, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Prospec recombinant wild type human prongf
    Stimulation of <t>GCs</t> with <t>proNGF</t> and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p
    Recombinant Wild Type Human Prongf, supplied by Prospec, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyvinylidene fluoride membranes
    Stimulation of <t>GCs</t> with <t>proNGF</t> and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p
    Polyvinylidene Fluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs prongf
    Minocycline inhibits <t>proNGF</t> expression after SCI. A , B , <t>NGF</t> mRNA ( A ) and protein expression ( B ) were increased after SCI. B , C , Anti-mature-NGF antibody detected both mature (14 kDa) and proNGF (26 kDa) ( B ), and anti-proNGF antibody detected the high-molecular-weight band of 26 kDa ( C ). Note that the extent of induction of proNGF was greater than that observed with mature NGF ( B ). D , Minocycline treatment decreased the level of proNGF expression compared with that observed in vehicle-treated control at 5 d after injury. E , Quantitative analysis of Western blots shows that minocycline significantly inhibited proNGF expression when compared with that in vehicle control at 5 d after injury. Values are mean ± SD of three separate experiments. * p
    Prongf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Biosensis prongf
    SPR analysis—calibration curves obtained with <t>proNGF</t> and <t>NGF</t> over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.
    Prongf, supplied by Biosensis, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Alomone Labs non cleavable prongf
    SPR analysis—calibration curves obtained with <t>proNGF</t> and <t>NGF</t> over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.
    Non Cleavable Prongf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore γ secretase inhibitor
    SPR analysis—calibration curves obtained with <t>proNGF</t> and <t>NGF</t> over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.
    γ Secretase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sf21 cells
    Western blot analysis of different mutants of proNGF. <t>Sf21</t> insect cells were stably transfected with mutated proNGF cDNA subcloned into pIZT vector. Mutant protein was secreted into the expression medium and purified by using either weak cation-exchange
    Sf21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA ep1318y antibodies
    Western blot analysis of different mutants of proNGF. <t>Sf21</t> insect cells were stably transfected with mutated proNGF cDNA subcloned into pIZT vector. Mutant protein was secreted into the expression medium and purified by using either weak cation-exchange
    Ep1318y Antibodies, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti gfap
    <t>EC-proNGF</t> overexpression did not cause neuronal toxicity or Muller cell activation. (a b) Representative images and quantitative analysis of the number of TUNEL-positive cells (green, arrows) in retina sections counterstained with Propedium Iodide (red). There were no significant changes in TUNEL-positive cells among examined groups including Cre-proNGF, Cre and proNGF, (n = 4). GCL; ganglion cell layer, INL; inner nuclear layer and ONL; outer nuclear layer. (c) Representative images of ganglion cell layer showing total number of cells stained with DAPI (blue), retinal ganglion cells (RGC) stained with Brn3A (Red) and the merge. (d) One way ANOVA Statistical analysis showed no significant change in the total number of neuronal cells or RGC count in GCL among all groups (n = 4–5). (e) Representative images of retinal sections stained with <t>GFAP,</t> marker for glial Muller activation showed no marked glial activation in Cre-proNGF compared to Cre or WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)
    Anti Gfap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies glial fibrillary acidic protein
    <t>EC-proNGF</t> overexpression did not cause neuronal toxicity or Muller cell activation. (a b) Representative images and quantitative analysis of the number of TUNEL-positive cells (green, arrows) in retina sections counterstained with Propedium Iodide (red). There were no significant changes in TUNEL-positive cells among examined groups including Cre-proNGF, Cre and proNGF, (n = 4). GCL; ganglion cell layer, INL; inner nuclear layer and ONL; outer nuclear layer. (c) Representative images of ganglion cell layer showing total number of cells stained with DAPI (blue), retinal ganglion cells (RGC) stained with Brn3A (Red) and the merge. (d) One way ANOVA Statistical analysis showed no significant change in the total number of neuronal cells or RGC count in GCL among all groups (n = 4–5). (e) Representative images of retinal sections stained with <t>GFAP,</t> marker for glial Muller activation showed no marked glial activation in Cre-proNGF compared to Cre or WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)
    Glial Fibrillary Acidic Protein, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 2723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 594
    <t>EC-proNGF</t> overexpression did not cause neuronal toxicity or Muller cell activation. (a b) Representative images and quantitative analysis of the number of TUNEL-positive cells (green, arrows) in retina sections counterstained with Propedium Iodide (red). There were no significant changes in TUNEL-positive cells among examined groups including Cre-proNGF, Cre and proNGF, (n = 4). GCL; ganglion cell layer, INL; inner nuclear layer and ONL; outer nuclear layer. (c) Representative images of ganglion cell layer showing total number of cells stained with DAPI (blue), retinal ganglion cells (RGC) stained with Brn3A (Red) and the merge. (d) One way ANOVA Statistical analysis showed no significant change in the total number of neuronal cells or RGC count in GCL among all groups (n = 4–5). (e) Representative images of retinal sections stained with <t>GFAP,</t> marker for glial Muller activation showed no marked glial activation in Cre-proNGF compared to Cre or WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)
    Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore human recombinant ngf
    <t>NGF</t> protein expression in cardiac ganglia from sham-operated (A, C) and stellate ganglionectomized (B, D) rats. A: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of large control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. B: After sympathectomy, although mature NGFβ-ir (white arrowhead) neurons are observed, the frequency of mature NGFβ-negative (filled arrowheads) neurons is increased. C: <t>ProNGF-ir</t> (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowhead). D: Following sympathectomy, neurons unstained for ProNGF (filled arrowheads) are increased within cardiac ganglia. E: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in D is 40μm.
    Human Recombinant Ngf, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bicinchoninic acid assay
    <t>NGF</t> protein expression in cardiac ganglia from sham-operated (A, C) and stellate ganglionectomized (B, D) rats. A: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of large control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. B: After sympathectomy, although mature NGFβ-ir (white arrowhead) neurons are observed, the frequency of mature NGFβ-negative (filled arrowheads) neurons is increased. C: <t>ProNGF-ir</t> (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowhead). D: Following sympathectomy, neurons unstained for ProNGF (filled arrowheads) are increased within cardiac ganglia. E: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in D is 40μm.
    Bicinchoninic Acid Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18662 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc igg
    PDR-aqueous humor stimulates tube-like structures in a <t>proNGF-dependent</t> manner. HRE cells were grown into confluence then trypsinized and mixed with reduced-growth factor Matrigel and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit <t>IgG</t> (1 μ g/mL). Representative micrographs for alignment of HRE into tube-like structures are shown after 18 hrs of incubation. Statistical analysis of tube length showed that aqueous humor increased mean tube formation 1.7-fold compared to the control group. Addition of anti-proNGF antibodies to aqueous humor samples significantly reduced the relative mean tube length but did not affect control group. Prior treatment of humor samples with rabbit IgG did not significantly reduce relative mean length when compared to the untreated aq. humor group ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P
    Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Epitomics p75ntr
    Immunostaining of NGF-β, <t>p75NTR</t> and trkA in uteri of ICR mice with adenomyosis or not . A: Uterus of 140 ± 5 d control ICR mice stained for NGF-β. B-E: Immnostaining of NGF-β in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. F: Uterus of 140 ± 5 d control ICR mice stained for p75NTR. G-J: Immnostaining of p75NTR in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. K-L: trkA immnoreactivity was detected in nerve fibers (K; arrows, nerve fibers), luminal and glandular epithelial cells of endometrium (L) in uterus of adenomyosis ICR mice aged 140 ± 5 d. (Bar = 100 μm).
    P75ntr, supplied by Epitomics, used in various techniques. Bioz Stars score: 89/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gfp
    Characterization of <t>proNGF</t> and <t>GFP</t> expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.
    Anti Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p75 neun
    Characterization of <t>proNGF</t> and <t>GFP</t> expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.
    P75 Neun, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam p75ntr
    Characterization of <t>proNGF</t> and <t>GFP</t> expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.
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    Vector Laboratories dab peroxidase hrp substrate kit
    Characterization of <t>proNGF</t> and <t>GFP</t> expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.
    Dab Peroxidase Hrp Substrate Kit, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Accurate Chemical & Scientific Corporation ox42
    Double immunofluorescence staining revealed that proNGF + cells (FITC, green) were reactive for NeuN (CY3, red) (A~D), GFAP (CY3, E~H) and <t>OX42</t> (CY3, I~L). Scale bars in A~D, I~L=50μm, E~H=100μm.
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    Image Search Results


    Histological localization of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in parenchymal hepatocytes of human livers with hepatolithiasis. Liver sections of lithiasis and contralateral parts in hepatolithiasis liver tissues ( n =5) and paralesional sections of hepatocellular carcinoma livers ( n =4) as non-hepatolithiasis control (NHC) underwent formalin-fixation, tissue-processing and paraffin-embedding procedures. Tissue sections were subjected to immunohistochemical staining for NGF, proNGF, TrkA and p75NTR. Scale bars=100 μm. Arrows indicate characteristic fatty changes in lithiasis tissues, while white and black arrowheads indicate immunoreactive signals in the hepatocytes and the infiltrated cells, respectively.

    Journal: Experimental & Molecular Medicine

    Article Title: Nerve growth factor upregulates sirtuin 1 expression in cholestasis: a potential therapeutic target

    doi: 10.1038/emm.2017.235

    Figure Lengend Snippet: Histological localization of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in parenchymal hepatocytes of human livers with hepatolithiasis. Liver sections of lithiasis and contralateral parts in hepatolithiasis liver tissues ( n =5) and paralesional sections of hepatocellular carcinoma livers ( n =4) as non-hepatolithiasis control (NHC) underwent formalin-fixation, tissue-processing and paraffin-embedding procedures. Tissue sections were subjected to immunohistochemical staining for NGF, proNGF, TrkA and p75NTR. Scale bars=100 μm. Arrows indicate characteristic fatty changes in lithiasis tissues, while white and black arrowheads indicate immunoreactive signals in the hepatocytes and the infiltrated cells, respectively.

    Article Snippet: Recombinant NGF and proNGF peptides were purchased from AbD Serotec (PMP04Z; Oxford, UK) and Alonmone Labs (N-250; Jerusalem, Israel), respectively.

    Techniques: Immunohistochemistry, Staining

    Expression of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in human livers with hepatolithiasis. ( a ) Hepatolithiasis samples ( n =5) included lithiasis and contralateral liver tissues. Paralesional protein extracts from patients with hepatocellular carcinoma were the non-hepatolithiasis controls (NHC, n , respectively. Densitometrical analyses of the expression levels of NGF and proNGF ( b ), as well as those of TrkA and p75NTR ( c ), are shown as the mean±s.e.m. of their ratios to internal actin controls. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Nerve growth factor upregulates sirtuin 1 expression in cholestasis: a potential therapeutic target

    doi: 10.1038/emm.2017.235

    Figure Lengend Snippet: Expression of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in human livers with hepatolithiasis. ( a ) Hepatolithiasis samples ( n =5) included lithiasis and contralateral liver tissues. Paralesional protein extracts from patients with hepatocellular carcinoma were the non-hepatolithiasis controls (NHC, n , respectively. Densitometrical analyses of the expression levels of NGF and proNGF ( b ), as well as those of TrkA and p75NTR ( c ), are shown as the mean±s.e.m. of their ratios to internal actin controls. * P

    Article Snippet: Recombinant NGF and proNGF peptides were purchased from AbD Serotec (PMP04Z; Oxford, UK) and Alonmone Labs (N-250; Jerusalem, Israel), respectively.

    Techniques: Expressing

    WT-ProNGF induces TrkA-dependent signaling cascades. PC12 cells ( panels A and B ) or PC12nnr5 cells ( panels C and D ) were incubated in DMEB for 1 h and then treated with 25 ng/ml NGF or 50 ng/ml WT-proNGF for the time points indicated. Cells were lysed

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: WT-ProNGF induces TrkA-dependent signaling cascades. PC12 cells ( panels A and B ) or PC12nnr5 cells ( panels C and D ) were incubated in DMEB for 1 h and then treated with 25 ng/ml NGF or 50 ng/ml WT-proNGF for the time points indicated. Cells were lysed

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques: Incubation

    ProNGF cleavage is required for TrkA activation. A , PC12 cells in DMEB were pretreated with dec-CMK (50 μ m ) for 30 min and then exposed to NGF (25 ng/ml) or WT-proNGF (50 ng/ml). TrkA was immunoprecipitated and phosphotyrosine levels were examined

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: ProNGF cleavage is required for TrkA activation. A , PC12 cells in DMEB were pretreated with dec-CMK (50 μ m ) for 30 min and then exposed to NGF (25 ng/ml) or WT-proNGF (50 ng/ml). TrkA was immunoprecipitated and phosphotyrosine levels were examined

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques: Activation Assay, Immunoprecipitation

    Inhibiting metalloproteases does not block proNGF-induced activation of TrkA signaling cascades. A , PC12 cells in DMEB were exposed to BB94 (10 μ m ) for 1 h, then they were treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the times indicated,

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: Inhibiting metalloproteases does not block proNGF-induced activation of TrkA signaling cascades. A , PC12 cells in DMEB were exposed to BB94 (10 μ m ) for 1 h, then they were treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the times indicated,

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques: Blocking Assay, Activation Assay

    WT-ProNGF induces robust TrkA phosphorylation and neurite outgrowth. A , PC12 cells were incubated in DMEB for 1 h then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the indicated times, then lysed, and TrkA was immunoprecipitated. Immunoblots

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: WT-ProNGF induces robust TrkA phosphorylation and neurite outgrowth. A , PC12 cells were incubated in DMEB for 1 h then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the indicated times, then lysed, and TrkA was immunoprecipitated. Immunoblots

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques: Incubation, Immunoprecipitation, Western Blot

    Recombinant proNGF is expressed and secreted in intact form. A , schematic representation of recombinant proNGF proteins used in this study. Position-1 refers to the cleavage site that generates fully processed NGF. AP-proNGF contains an amino-terminal

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: Recombinant proNGF is expressed and secreted in intact form. A , schematic representation of recombinant proNGF proteins used in this study. Position-1 refers to the cleavage site that generates fully processed NGF. AP-proNGF contains an amino-terminal

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques: Recombinant

    Endocytosis is required for WT-proNGF treatment to induce TrkA signaling. A and B , PC12 cells in DMEB were exposed to 0.45 m sucrose for 15 min and then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for 10 or 30 min, as indicated. A , cells were

    Journal: The Journal of Biological Chemistry

    Article Title: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation *Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation * S⃞

    doi: 10.1074/jbc.M710018200

    Figure Lengend Snippet: Endocytosis is required for WT-proNGF treatment to induce TrkA signaling. A and B , PC12 cells in DMEB were exposed to 0.45 m sucrose for 15 min and then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for 10 or 30 min, as indicated. A , cells were

    Article Snippet: To confirm that these distinct proNGF proteins were intact and to confirm relative concentrations of these proteins, they were evaluated by immunoblotting using an antibody directed against mature NGF. shows that recombinant AP-proNGF, WT-proNGF, and proNGF123 are intact and therefore appropriate for use in cell-based assays.

    Techniques:

    SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

    Article Snippet: The primary antibody used was anti-proNGF (PAb Chemicon Millipore) and the secondary antibody was goat anti-rabbit HRP-conjugated (Jackson).

    Techniques: SPR Assay

    Analysis of transgenic and wild-type mice samples by SPR . Injection of samples from transgenic and from WT mice over the FPro10 anti-proNGF antibody (A) and over the Millipore anti-proNGF antibody (B) (blank subtracted curves). Representation of the SPR curves after subtraction of the WT mice signals. Curve of TgproNGF#72 mice subtracted the curve of WT for the FPro10 anti-proNGF antibody ( A —insert), and for the Millipore anti-proNGF antibody ( B —insert). In all panels: Red curve: TgproNGF#72, blue curve: WT.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: Analysis of transgenic and wild-type mice samples by SPR . Injection of samples from transgenic and from WT mice over the FPro10 anti-proNGF antibody (A) and over the Millipore anti-proNGF antibody (B) (blank subtracted curves). Representation of the SPR curves after subtraction of the WT mice signals. Curve of TgproNGF#72 mice subtracted the curve of WT for the FPro10 anti-proNGF antibody ( A —insert), and for the Millipore anti-proNGF antibody ( B —insert). In all panels: Red curve: TgproNGF#72, blue curve: WT.

    Article Snippet: The primary antibody used was anti-proNGF (PAb Chemicon Millipore) and the secondary antibody was goat anti-rabbit HRP-conjugated (Jackson).

    Techniques: Transgenic Assay, Mouse Assay, SPR Assay, Injection

    ELISA for the differential detection of NGF and ProNGF . (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988 ) on a solid support. Subsequent detection of proNGF by traditional sandwich ELISA. Antibodies tested in different combinations: Anti-NGF pAb H20 (Santa Cruz no. sc-548), Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-NGF pAb (Sigma no. N 6655), Anti-proNGF pAb (Sigma no. P 5498), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF mAb 256 (R D no. MAB256). (B) Strategy: capture proNGF and measure proNGF (Exploiting anti—proNGF antibodies in ELISA sandwich). Antibodies tested in different combinations: Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-proNGF Novus (no. S-080-100), Anti-NGF mAb 256 (R D no. MAB256), Anti-proNGF mAb (clone EP1318Y) (Millipore no. 04-1142), Anti-proNGF pAb Chemicon (Millipore no. AB9040), Anti-proNGF pAb Alomone (no. ANT-005), Anti-NGF Abnova (no. PAB0755).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: ELISA for the differential detection of NGF and ProNGF . (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988 ) on a solid support. Subsequent detection of proNGF by traditional sandwich ELISA. Antibodies tested in different combinations: Anti-NGF pAb H20 (Santa Cruz no. sc-548), Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-NGF pAb (Sigma no. N 6655), Anti-proNGF pAb (Sigma no. P 5498), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF mAb 256 (R D no. MAB256). (B) Strategy: capture proNGF and measure proNGF (Exploiting anti—proNGF antibodies in ELISA sandwich). Antibodies tested in different combinations: Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-proNGF Novus (no. S-080-100), Anti-NGF mAb 256 (R D no. MAB256), Anti-proNGF mAb (clone EP1318Y) (Millipore no. 04-1142), Anti-proNGF pAb Chemicon (Millipore no. AB9040), Anti-proNGF pAb Alomone (no. ANT-005), Anti-NGF Abnova (no. PAB0755).

    Article Snippet: The primary antibody used was anti-proNGF (PAb Chemicon Millipore) and the secondary antibody was goat anti-rabbit HRP-conjugated (Jackson).

    Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

    Binding analysis of NGF and proNGF mixtures to different antibodies . The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (segmented blue line, theoretical value); 40 nM proNGF + 20 nM NGF (solid purple line, experimental value); 40 nM proNGF (solid orange line, experimental value); 40 nM proNGF + 20 nM NGF (segmented purple line, theoretical value); 20 nM proNGF + 20 nM NGF (solid green line, experimental value); 20 nM proNGF (solid red line, experimental value); 20 nM proNGF + 20 nM NGF (segmented green line, theoretical value); 20 nM NGF (solid dark blue line, experimental value). The segmented lines represents the theoretical curves for the point-to-point algebraic sum of the experimental curves of the single components, at the concentrations indicated.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: Binding analysis of NGF and proNGF mixtures to different antibodies . The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (segmented blue line, theoretical value); 40 nM proNGF + 20 nM NGF (solid purple line, experimental value); 40 nM proNGF (solid orange line, experimental value); 40 nM proNGF + 20 nM NGF (segmented purple line, theoretical value); 20 nM proNGF + 20 nM NGF (solid green line, experimental value); 20 nM proNGF (solid red line, experimental value); 20 nM proNGF + 20 nM NGF (segmented green line, theoretical value); 20 nM NGF (solid dark blue line, experimental value). The segmented lines represents the theoretical curves for the point-to-point algebraic sum of the experimental curves of the single components, at the concentrations indicated.

    Article Snippet: The primary antibody used was anti-proNGF (PAb Chemicon Millipore) and the secondary antibody was goat anti-rabbit HRP-conjugated (Jackson).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, SPR Assay

    IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. ( 2013 ). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. ( 2013 ). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

    Article Snippet: The primary antibody used was anti-proNGF (PAb Chemicon Millipore) and the secondary antibody was goat anti-rabbit HRP-conjugated (Jackson).

    Techniques: Western Blot, Transgenic Assay, Mouse Assay, Mass Spectrometry, Recombinant

    NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) . The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t -student test was carried out and the p -values were calculated. Two asterisks on the histograms mean a p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) . The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t -student test was carried out and the p -values were calculated. Two asterisks on the histograms mean a p

    Article Snippet: While for the anti-proNGF antibodies (mAb FPro10 and mAb Millipore) this effect is negligible (Table ), the interference effect was more significant for the anti-NGF mAb αD11 (Table ).

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Expression and localization of p75 NTR and neurotoxic factors increase during the progression of DR. The experimental paradigm and the endpoints of the mouse DR model are shown. Drug treatments were injected intravitreally at week 2.5 of diabetes in this paradigm. A , p75 NTR , proNGF, TNFα, and α 2 M expression in whole mice retina analyzed by Western blot from 1–6 weeks after induction of diabetes. There were increases of p75 NTR during week 1, of proNGF during week 3, of TNFα during week 4, and of α 2 M during week 5. B , Quantification. * p

    Journal: The Journal of Neuroscience

    Article Title: p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

    doi: 10.1523/JNEUROSCI.4278-15.2016

    Figure Lengend Snippet: Expression and localization of p75 NTR and neurotoxic factors increase during the progression of DR. The experimental paradigm and the endpoints of the mouse DR model are shown. Drug treatments were injected intravitreally at week 2.5 of diabetes in this paradigm. A , p75 NTR , proNGF, TNFα, and α 2 M expression in whole mice retina analyzed by Western blot from 1–6 weeks after induction of diabetes. There were increases of p75 NTR during week 1, of proNGF during week 3, of TNFα during week 4, and of α 2 M during week 5. B , Quantification. * p

    Article Snippet: Treatment of rMC-1 cells for 24 h with recombinant proNGF caused increases in TNFα mRNA, in a dose-dependent manner ( B ).

    Techniques: Expressing, Injection, Mouse Assay, Western Blot

    Pharmacological inhibition of p75 NTR or proNGF prevent elevation of TNFα and α 2 M in DR. The levels of TNFα transcript were quantified by quantitative real-time PCR. A , Retinas from 6 week diabetic mice ± treatment at week 2.5 of diabetes with anti-proNGF mAb, or THX-B, or vehicle, compared with age-matched naive control animals ( n = 3/group). ** p

    Journal: The Journal of Neuroscience

    Article Title: p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

    doi: 10.1523/JNEUROSCI.4278-15.2016

    Figure Lengend Snippet: Pharmacological inhibition of p75 NTR or proNGF prevent elevation of TNFα and α 2 M in DR. The levels of TNFα transcript were quantified by quantitative real-time PCR. A , Retinas from 6 week diabetic mice ± treatment at week 2.5 of diabetes with anti-proNGF mAb, or THX-B, or vehicle, compared with age-matched naive control animals ( n = 3/group). ** p

    Article Snippet: Treatment of rMC-1 cells for 24 h with recombinant proNGF caused increases in TNFα mRNA, in a dose-dependent manner ( B ).

    Techniques: Inhibition, Real-time Polymerase Chain Reaction, Mouse Assay

    Inhibition of p75 NTR or proNGF efficiently reduces pathological vascular permeability in DR. Quantification of the levels of Sema3A transcript by qPCR on ( A ) Retinal neuronal cell line (RGC 5) treated for 2, 4, and 8 h with TNFα. Sema3A increased over time, compared with basal levels. *** p

    Journal: The Journal of Neuroscience

    Article Title: p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

    doi: 10.1523/JNEUROSCI.4278-15.2016

    Figure Lengend Snippet: Inhibition of p75 NTR or proNGF efficiently reduces pathological vascular permeability in DR. Quantification of the levels of Sema3A transcript by qPCR on ( A ) Retinal neuronal cell line (RGC 5) treated for 2, 4, and 8 h with TNFα. Sema3A increased over time, compared with basal levels. *** p

    Article Snippet: Treatment of rMC-1 cells for 24 h with recombinant proNGF caused increases in TNFα mRNA, in a dose-dependent manner ( B ).

    Techniques: Inhibition, Permeability, Real-time Polymerase Chain Reaction

    Model summarizing the etiology of p75 NTR in DR. In a healthy mature retina, the levels of p75 NTR are low. In diabetes, proNGF is significantly increased and activates p75 NTR , which is upregulated in Müller glial cells and pericytes. In Müller cells, p75 NTR activity triggers the expression/secretion of α 2 M and TNFα. α 2 M potentiates proNGF, thus creating a vicious cycle. Upregulated TNFα binds to receptors in RGCs inducing the release of Sema3A and eventual RGC death. Sema3A binds to its receptor Nrp1 and provokes loosening of endothelial cell junctions in the BRB and leads to plasma extravasation. In pericytes, p75 NTR activity could presumably trigger dysfunction and loss of vascular integrity, but this was not evaluated in this study. Intravitreal injections of THX-B or the anti-proNGF mAb in diabetic mice significantly diminished Müller glia activation, decreased TNFα and α 2 M expression, limited Sema3A production, maintained BRB integrity, and prevented RGC neuronal cell death.

    Journal: The Journal of Neuroscience

    Article Title: p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

    doi: 10.1523/JNEUROSCI.4278-15.2016

    Figure Lengend Snippet: Model summarizing the etiology of p75 NTR in DR. In a healthy mature retina, the levels of p75 NTR are low. In diabetes, proNGF is significantly increased and activates p75 NTR , which is upregulated in Müller glial cells and pericytes. In Müller cells, p75 NTR activity triggers the expression/secretion of α 2 M and TNFα. α 2 M potentiates proNGF, thus creating a vicious cycle. Upregulated TNFα binds to receptors in RGCs inducing the release of Sema3A and eventual RGC death. Sema3A binds to its receptor Nrp1 and provokes loosening of endothelial cell junctions in the BRB and leads to plasma extravasation. In pericytes, p75 NTR activity could presumably trigger dysfunction and loss of vascular integrity, but this was not evaluated in this study. Intravitreal injections of THX-B or the anti-proNGF mAb in diabetic mice significantly diminished Müller glia activation, decreased TNFα and α 2 M expression, limited Sema3A production, maintained BRB integrity, and prevented RGC neuronal cell death.

    Article Snippet: Treatment of rMC-1 cells for 24 h with recombinant proNGF caused increases in TNFα mRNA, in a dose-dependent manner ( B ).

    Techniques: Activity Assay, Expressing, Mouse Assay, Activation Assay

    Changes in adipose tissue expression of A) pro nerve growth factor (proNGF), B) mature NGF (mNGF) and C) the mNGF/proNGF ratio after 5 weeks of electroacupuncture (EA) measured by ELISA. D) Representative blot of each proNGF splicing variant from before and after 5 weeks of EA measured by western blot and corresponding densitometry analyses for E) proNGF 50 kDa; F) proNGF 34 kDa and G) all proNGF variants detectable in adipose tissue, and H) representative western blots and corresponding gel densitometry analyses for adipose tissue expression of the mNGF/proNGF receptors I) phosphorylated‐TrkA and J) p75 NTR .

    Journal: Obesity Science & Practice

    Article Title: Changes in HbA1c and circulating and adipose tissue androgen levels in overweight‐obese women with polycystic ovary syndrome in response to electroacupuncture) Changes in HbA1c and circulating and adipose tissue androgen levels in overweight‐obese women with polycystic ovary syndrome in response to electroacupuncture

    doi: 10.1002/osp4.78

    Figure Lengend Snippet: Changes in adipose tissue expression of A) pro nerve growth factor (proNGF), B) mature NGF (mNGF) and C) the mNGF/proNGF ratio after 5 weeks of electroacupuncture (EA) measured by ELISA. D) Representative blot of each proNGF splicing variant from before and after 5 weeks of EA measured by western blot and corresponding densitometry analyses for E) proNGF 50 kDa; F) proNGF 34 kDa and G) all proNGF variants detectable in adipose tissue, and H) representative western blots and corresponding gel densitometry analyses for adipose tissue expression of the mNGF/proNGF receptors I) phosphorylated‐TrkA and J) p75 NTR .

    Article Snippet: Adipose tissue protein concentration of mature mNGF and proNGF were detected and quantified using MAB5260Z clone 27/21 and EP1318Y antibodies (Merck Millipore), respectively, in a homemade sandwich ELISA device.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Variant Assay, Western Blot

    Stimulation of GCs with proNGF and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p

    Journal: Hormone molecular biology and clinical investigation

    Article Title: Pro-nerve growth factor in the ovary and human granulosa cells

    doi: 10.1515/hmbci-2015-0028

    Figure Lengend Snippet: Stimulation of GCs with proNGF and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E) A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p

    Article Snippet: Human GCs were incubated with recombinant proNGF (Cedarlane, Burlington, Canada) and recombinant NGF (Alomone Labs, Jerusalem, Israel) each with 50 ng/ml for 1 h or 24 h in DMEM/Ham's F12 media without FCS, to examine downstream effects on early growth response 1 (EGR1) and choline acetyl-transferase (CHAT) level exerted by the factors.

    Techniques: Positive Control, Recombinant

    ProNGF and NGF in human GCs Left panel: Example of a Western blot: ProNGF is detected in human GCs (cells cultured for 2 and 3 days are shown); middle panel: Control blot, in which pre-adsorbed anti-proNGF antiserum was used; right panel: Example of a Western blot, in which an antiserum to NGF was used. Note that this antiserum, as expected, recognizes both NGF and proNGF and that the GCs shown contain abundant proNGF, while NGF was much less abundant.

    Journal: Hormone molecular biology and clinical investigation

    Article Title: Pro-nerve growth factor in the ovary and human granulosa cells

    doi: 10.1515/hmbci-2015-0028

    Figure Lengend Snippet: ProNGF and NGF in human GCs Left panel: Example of a Western blot: ProNGF is detected in human GCs (cells cultured for 2 and 3 days are shown); middle panel: Control blot, in which pre-adsorbed anti-proNGF antiserum was used; right panel: Example of a Western blot, in which an antiserum to NGF was used. Note that this antiserum, as expected, recognizes both NGF and proNGF and that the GCs shown contain abundant proNGF, while NGF was much less abundant.

    Article Snippet: Human GCs were incubated with recombinant proNGF (Cedarlane, Burlington, Canada) and recombinant NGF (Alomone Labs, Jerusalem, Israel) each with 50 ng/ml for 1 h or 24 h in DMEM/Ham's F12 media without FCS, to examine downstream effects on early growth response 1 (EGR1) and choline acetyl-transferase (CHAT) level exerted by the factors.

    Techniques: Western Blot, Cell Culture

    EGR1 and CHAT level increase in human GCs after stimulation with NGF A: Example of a Western blot with human GCs on day 2 of culture; stimulation of human GCs with NGF (50 ng/ml) for 1 h led to an increase in EGR1 levels whereas proNGF stimulation (50 ng/ml) did not. EGR1 pre-adsorption was used as control and β-actin as internal standard. B: Densitometric analysis of the experiments performed on day 2 of human GC culture. Each column represents the ratio of EGR1 to untreated control (mean ± SEM) of four independent experiments. *, p

    Journal: Hormone molecular biology and clinical investigation

    Article Title: Pro-nerve growth factor in the ovary and human granulosa cells

    doi: 10.1515/hmbci-2015-0028

    Figure Lengend Snippet: EGR1 and CHAT level increase in human GCs after stimulation with NGF A: Example of a Western blot with human GCs on day 2 of culture; stimulation of human GCs with NGF (50 ng/ml) for 1 h led to an increase in EGR1 levels whereas proNGF stimulation (50 ng/ml) did not. EGR1 pre-adsorption was used as control and β-actin as internal standard. B: Densitometric analysis of the experiments performed on day 2 of human GC culture. Each column represents the ratio of EGR1 to untreated control (mean ± SEM) of four independent experiments. *, p

    Article Snippet: Human GCs were incubated with recombinant proNGF (Cedarlane, Burlington, Canada) and recombinant NGF (Alomone Labs, Jerusalem, Israel) each with 50 ng/ml for 1 h or 24 h in DMEM/Ham's F12 media without FCS, to examine downstream effects on early growth response 1 (EGR1) and choline acetyl-transferase (CHAT) level exerted by the factors.

    Techniques: Western Blot, Adsorption

    The NGF precursor proNGF (A) and mature NGF (B) are present in the cells of the follicular wall in human ovaries Immunohistochemical staining of sections of human ovaries reveals that proNGF is present in cells of a large antral follicle, namely in GCs and theca cells (TCs). Positive staining reaction is indicated by the brown color. Note that the antiserum is specific for the pro-region of the proNGF molecule and does not recognize mature NGF. Bar: 30 μm. Insert control: Result of an experiment in which the proNGF antiserum was pre-adsorbed.

    Journal: Hormone molecular biology and clinical investigation

    Article Title: Pro-nerve growth factor in the ovary and human granulosa cells

    doi: 10.1515/hmbci-2015-0028

    Figure Lengend Snippet: The NGF precursor proNGF (A) and mature NGF (B) are present in the cells of the follicular wall in human ovaries Immunohistochemical staining of sections of human ovaries reveals that proNGF is present in cells of a large antral follicle, namely in GCs and theca cells (TCs). Positive staining reaction is indicated by the brown color. Note that the antiserum is specific for the pro-region of the proNGF molecule and does not recognize mature NGF. Bar: 30 μm. Insert control: Result of an experiment in which the proNGF antiserum was pre-adsorbed.

    Article Snippet: Human GCs were incubated with recombinant proNGF (Cedarlane, Burlington, Canada) and recombinant NGF (Alomone Labs, Jerusalem, Israel) each with 50 ng/ml for 1 h or 24 h in DMEM/Ham's F12 media without FCS, to examine downstream effects on early growth response 1 (EGR1) and choline acetyl-transferase (CHAT) level exerted by the factors.

    Techniques: Immunohistochemistry, Staining

    Minocycline inhibits proNGF expression after SCI. A , B , NGF mRNA ( A ) and protein expression ( B ) were increased after SCI. B , C , Anti-mature-NGF antibody detected both mature (14 kDa) and proNGF (26 kDa) ( B ), and anti-proNGF antibody detected the high-molecular-weight band of 26 kDa ( C ). Note that the extent of induction of proNGF was greater than that observed with mature NGF ( B ). D , Minocycline treatment decreased the level of proNGF expression compared with that observed in vehicle-treated control at 5 d after injury. E , Quantitative analysis of Western blots shows that minocycline significantly inhibited proNGF expression when compared with that in vehicle control at 5 d after injury. Values are mean ± SD of three separate experiments. * p

    Journal: The Journal of Neuroscience

    Article Title: Minocycline Alleviates Death of Oligodendrocytes by Inhibiting Pro-Nerve Growth Factor Production in Microglia after Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1661-07.2007

    Figure Lengend Snippet: Minocycline inhibits proNGF expression after SCI. A , B , NGF mRNA ( A ) and protein expression ( B ) were increased after SCI. B , C , Anti-mature-NGF antibody detected both mature (14 kDa) and proNGF (26 kDa) ( B ), and anti-proNGF antibody detected the high-molecular-weight band of 26 kDa ( C ). Note that the extent of induction of proNGF was greater than that observed with mature NGF ( B ). D , Minocycline treatment decreased the level of proNGF expression compared with that observed in vehicle-treated control at 5 d after injury. E , Quantitative analysis of Western blots shows that minocycline significantly inhibited proNGF expression when compared with that in vehicle control at 5 d after injury. Values are mean ± SD of three separate experiments. * p

    Article Snippet: The membranes were blocked with 5% nonfat skim milk in TBS for 1 h at room temperature and then incubated with polyclonal antibodies against p38MAPK, p-p38MAPK, MAPKAPK-2, p-MAPKAPK-2 (1:1000 dilution; Cell Signaling Technology), NGF (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), proNGF (1:1000 dilution; Alomone Labs), p75NTR (1:1000 dilution; Promega), and RhoA (1:1000 dilution; Santa Cruz Biotechnology).

    Techniques: Expressing, Molecular Weight, Western Blot

    A , Microglia-derived proNGF induces apoptosis of oligodendrocytes in culture. Immunocytochemical analysis shows that MBP-positive oligodendrocytes expressed p75 NTR (arrows). Scale bar, 20 μm. Differentiated oligodendrocytes were treated with LPS-treated BV2 cell culture medium. After 24 h, cells were processed for TUNEL and MBP staining. B , Representative photographs show that LPS-treated BV2 cell culture medium or recombinant NGF (as a positive control) induced the apoptotic cell death of oligodendrocytes as revealed by the presence of both TUNEL- and MBP-positive cells (arrows). Note that control shows no TUNEL/MBP-positive cells. Scale bar, 20 μm. C , Quantitative analyses of TUNEL-positive oligodendrocytes show that LPS-induced oligodendrocyte cell death was significantly inhibited by minocycline or SB203580 treatment. Also, oligodendrocyte cell death was significantly attenuated when LPS-treated BV2 cell culture medium subjected to immunoprecipitation using a neutralizing anti-NGF polyclonal antibody or when oligodendrocytes were treated with anti- p75 NTR antibody before treatment with LPS-treated BV2 cell culture medium ( C ). Values are mean ± SD of three separate experiments. * p

    Journal: The Journal of Neuroscience

    Article Title: Minocycline Alleviates Death of Oligodendrocytes by Inhibiting Pro-Nerve Growth Factor Production in Microglia after Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1661-07.2007

    Figure Lengend Snippet: A , Microglia-derived proNGF induces apoptosis of oligodendrocytes in culture. Immunocytochemical analysis shows that MBP-positive oligodendrocytes expressed p75 NTR (arrows). Scale bar, 20 μm. Differentiated oligodendrocytes were treated with LPS-treated BV2 cell culture medium. After 24 h, cells were processed for TUNEL and MBP staining. B , Representative photographs show that LPS-treated BV2 cell culture medium or recombinant NGF (as a positive control) induced the apoptotic cell death of oligodendrocytes as revealed by the presence of both TUNEL- and MBP-positive cells (arrows). Note that control shows no TUNEL/MBP-positive cells. Scale bar, 20 μm. C , Quantitative analyses of TUNEL-positive oligodendrocytes show that LPS-induced oligodendrocyte cell death was significantly inhibited by minocycline or SB203580 treatment. Also, oligodendrocyte cell death was significantly attenuated when LPS-treated BV2 cell culture medium subjected to immunoprecipitation using a neutralizing anti-NGF polyclonal antibody or when oligodendrocytes were treated with anti- p75 NTR antibody before treatment with LPS-treated BV2 cell culture medium ( C ). Values are mean ± SD of three separate experiments. * p

    Article Snippet: The membranes were blocked with 5% nonfat skim milk in TBS for 1 h at room temperature and then incubated with polyclonal antibodies against p38MAPK, p-p38MAPK, MAPKAPK-2, p-MAPKAPK-2 (1:1000 dilution; Cell Signaling Technology), NGF (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), proNGF (1:1000 dilution; Alomone Labs), p75NTR (1:1000 dilution; Promega), and RhoA (1:1000 dilution; Santa Cruz Biotechnology).

    Techniques: Derivative Assay, Cell Culture, TUNEL Assay, Staining, Recombinant, Positive Control, Immunoprecipitation

    Deletion of p75 NTR eliminates the diabetes-induced imbalance in proNGF and NGF expression. ( a ) Representative blots and statistical analysis of proNGF and ( b ) NGF protein levels in retinas of WT control (WTC), WT diabetic (WTD), HOC and HOD mice after

    Journal: Diabetologia

    Article Title: Modulation of p75NTR prevents diabetes- and proNGF-induced retinal inflammation and blood–retina barrier breakdown in mice and rats

    doi: 10.1007/s00125-013-2998-6

    Figure Lengend Snippet: Deletion of p75 NTR eliminates the diabetes-induced imbalance in proNGF and NGF expression. ( a ) Representative blots and statistical analysis of proNGF and ( b ) NGF protein levels in retinas of WT control (WTC), WT diabetic (WTD), HOC and HOD mice after

    Article Snippet: The following primary antibodies were used: polyclonal anti-NGF and anti-proNGF (Alomone Labs, Jerusalem, Israel); polyclonal anti-p75NTR (catalogue number 07–476), polyclonal anti-vascular endothelial growth factor (VEGF) and monoclonal anti-GFP antibody (Millipore, Billerica, MA, USA); monoclonal anti-TNF-α, polyclonal anti-IL-1β and polyclonal anti-sortilin (Abcam, Cambridge, MA, USA); monoclonal IgG2 (R & D Systems, Minneapolis, MN, USA); polyclonal anti-glial fibrillary acidic protein (GFAP) and monoclonal anti-vimentin (Thermo Fisher); polyclonal β-actin (Sigma-Aldrich, St Louis, MO, USA); monoclonal anti-brain-specific homeobox/POU domain protein 3A (BRN3A) and polyclonal anti-NFκB-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and monoclonal anti-phospho-NFκB (p65 subunit) (Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Mouse Assay

    SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: SPR Assay

    AlphaLISA experiments: curves of NGF, proNGF, mix of NGF + proNGF, comparison at different incubation times . Curves of NGF, proNGF, mix of NGF + proNGF, linearly interpolated. NGF and proNGF were assayed separately and together (dynamic range: 2000–4 pg/ml, 1:2 dilutions, in duplicates), in different stoichiometric ratio. The dilution were done in AlphaLISA NaCl buffer (Perkin Elmer). The protocol is described in the methods sections and called for two incubation times. In (A) the first incubation time lasted 60 min and the second one 30 min, as suggested by the manufacturer's protocol. In (B) , a comparison between different incubation times is shown. The first incubation time was 15, 30, or 60 min, while the second one was 20 min for all the curves. The signal was read on the Perkin Elmer instrument EnVision®.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: AlphaLISA experiments: curves of NGF, proNGF, mix of NGF + proNGF, comparison at different incubation times . Curves of NGF, proNGF, mix of NGF + proNGF, linearly interpolated. NGF and proNGF were assayed separately and together (dynamic range: 2000–4 pg/ml, 1:2 dilutions, in duplicates), in different stoichiometric ratio. The dilution were done in AlphaLISA NaCl buffer (Perkin Elmer). The protocol is described in the methods sections and called for two incubation times. In (A) the first incubation time lasted 60 min and the second one 30 min, as suggested by the manufacturer's protocol. In (B) , a comparison between different incubation times is shown. The first incubation time was 15, 30, or 60 min, while the second one was 20 min for all the curves. The signal was read on the Perkin Elmer instrument EnVision®.

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: Incubation

    NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) . The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t -student test was carried out and the p -values were calculated. Two asterisks on the histograms mean a p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) . The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t -student test was carried out and the p -values were calculated. Two asterisks on the histograms mean a p

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

    ELISA for the differential detection of NGF and ProNGF . (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988 ) on a solid support. Subsequent detection of proNGF by traditional sandwich ELISA. Antibodies tested in different combinations: Anti-NGF pAb H20 (Santa Cruz no. sc-548), Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-NGF pAb (Sigma no. N 6655), Anti-proNGF pAb (Sigma no. P 5498), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF mAb 256 (R D no. MAB256). (B) Strategy: capture proNGF and measure proNGF (Exploiting anti—proNGF antibodies in ELISA sandwich). Antibodies tested in different combinations: Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-proNGF Novus (no. S-080-100), Anti-NGF mAb 256 (R D no. MAB256), Anti-proNGF mAb (clone EP1318Y) (Millipore no. 04-1142), Anti-proNGF pAb Chemicon (Millipore no. AB9040), Anti-proNGF pAb Alomone (no. ANT-005), Anti-NGF Abnova (no. PAB0755).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: ELISA for the differential detection of NGF and ProNGF . (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988 ) on a solid support. Subsequent detection of proNGF by traditional sandwich ELISA. Antibodies tested in different combinations: Anti-NGF pAb H20 (Santa Cruz no. sc-548), Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-NGF pAb (Sigma no. N 6655), Anti-proNGF pAb (Sigma no. P 5498), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF mAb 256 (R D no. MAB256). (B) Strategy: capture proNGF and measure proNGF (Exploiting anti—proNGF antibodies in ELISA sandwich). Antibodies tested in different combinations: Anti-proNGF scFV FPro10 (Paoletti et al., 2012 ), mAb αD11 (Cattaneo et al., 1988 ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-proNGF Novus (no. S-080-100), Anti-NGF mAb 256 (R D no. MAB256), Anti-proNGF mAb (clone EP1318Y) (Millipore no. 04-1142), Anti-proNGF pAb Chemicon (Millipore no. AB9040), Anti-proNGF pAb Alomone (no. ANT-005), Anti-NGF Abnova (no. PAB0755).

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

    Binding analysis of NGF and proNGF mixtures to different antibodies . The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (segmented blue line, theoretical value); 40 nM proNGF + 20 nM NGF (solid purple line, experimental value); 40 nM proNGF (solid orange line, experimental value); 40 nM proNGF + 20 nM NGF (segmented purple line, theoretical value); 20 nM proNGF + 20 nM NGF (solid green line, experimental value); 20 nM proNGF (solid red line, experimental value); 20 nM proNGF + 20 nM NGF (segmented green line, theoretical value); 20 nM NGF (solid dark blue line, experimental value). The segmented lines represents the theoretical curves for the point-to-point algebraic sum of the experimental curves of the single components, at the concentrations indicated.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: Binding analysis of NGF and proNGF mixtures to different antibodies . The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (segmented blue line, theoretical value); 40 nM proNGF + 20 nM NGF (solid purple line, experimental value); 40 nM proNGF (solid orange line, experimental value); 40 nM proNGF + 20 nM NGF (segmented purple line, theoretical value); 20 nM proNGF + 20 nM NGF (solid green line, experimental value); 20 nM proNGF (solid red line, experimental value); 20 nM proNGF + 20 nM NGF (segmented green line, theoretical value); 20 nM NGF (solid dark blue line, experimental value). The segmented lines represents the theoretical curves for the point-to-point algebraic sum of the experimental curves of the single components, at the concentrations indicated.

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, SPR Assay

    IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. ( 2013 ). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    doi: 10.3389/fnmol.2016.00063

    Figure Lengend Snippet: IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. ( 2013 ). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

    Article Snippet: The possible cross-reactivity with proNGF is reported in the NGF Biosensis datasheet, but is declared to be unimportant, since it is only observed at high protein concentration.

    Techniques: Western Blot, Transgenic Assay, Mouse Assay, Mass Spectrometry, Recombinant

    Western blot analysis of different mutants of proNGF. Sf21 insect cells were stably transfected with mutated proNGF cDNA subcloned into pIZT vector. Mutant protein was secreted into the expression medium and purified by using either weak cation-exchange

    Journal:

    Article Title: Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization

    doi: 10.1073/pnas.0604139103

    Figure Lengend Snippet: Western blot analysis of different mutants of proNGF. Sf21 insect cells were stably transfected with mutated proNGF cDNA subcloned into pIZT vector. Mutant protein was secreted into the expression medium and purified by using either weak cation-exchange

    Article Snippet: Stable transfectants of Sf21 cells were generated by transforming the cells with pIZT vector, which contained mutated proNGF cDNAs, by using lipofectamine reagent (Invitrogen) according to the manufacturer's instructions.

    Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Purification

    EC-proNGF overexpression did not cause neuronal toxicity or Muller cell activation. (a b) Representative images and quantitative analysis of the number of TUNEL-positive cells (green, arrows) in retina sections counterstained with Propedium Iodide (red). There were no significant changes in TUNEL-positive cells among examined groups including Cre-proNGF, Cre and proNGF, (n = 4). GCL; ganglion cell layer, INL; inner nuclear layer and ONL; outer nuclear layer. (c) Representative images of ganglion cell layer showing total number of cells stained with DAPI (blue), retinal ganglion cells (RGC) stained with Brn3A (Red) and the merge. (d) One way ANOVA Statistical analysis showed no significant change in the total number of neuronal cells or RGC count in GCL among all groups (n = 4–5). (e) Representative images of retinal sections stained with GFAP, marker for glial Muller activation showed no marked glial activation in Cre-proNGF compared to Cre or WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

    doi: 10.1016/j.bbadis.2017.12.023

    Figure Lengend Snippet: EC-proNGF overexpression did not cause neuronal toxicity or Muller cell activation. (a b) Representative images and quantitative analysis of the number of TUNEL-positive cells (green, arrows) in retina sections counterstained with Propedium Iodide (red). There were no significant changes in TUNEL-positive cells among examined groups including Cre-proNGF, Cre and proNGF, (n = 4). GCL; ganglion cell layer, INL; inner nuclear layer and ONL; outer nuclear layer. (c) Representative images of ganglion cell layer showing total number of cells stained with DAPI (blue), retinal ganglion cells (RGC) stained with Brn3A (Red) and the merge. (d) One way ANOVA Statistical analysis showed no significant change in the total number of neuronal cells or RGC count in GCL among all groups (n = 4–5). (e) Representative images of retinal sections stained with GFAP, marker for glial Muller activation showed no marked glial activation in Cre-proNGF compared to Cre or WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)

    Article Snippet: After 4-weeks of proNGF expression, frozen retinal cryo-sections (10-μm thickness) were permealized, blocked in goat serum and incubated with primary antibodies including anti-GFP (Abcam, Rabbit polyclonal, 1:200), anti-proNGF (Alomone, Israel, Rabbit polyclonal, 1:100), anti-GFAP (polyclonal, Affinity Bioreagents, Rockford, IL, USA; 1:200), followed by Oregon-green or Texas-red conjugated goat antirabbit secondary antibody (Invitrogen, Carlsbad, CA, USA; 1:500).

    Techniques: Over Expression, Activation Assay, TUNEL Assay, Staining, Marker, Mouse Assay

    NGF protein expression in cardiac ganglia from sham-operated (A, C) and stellate ganglionectomized (B, D) rats. A: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of large control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. B: After sympathectomy, although mature NGFβ-ir (white arrowhead) neurons are observed, the frequency of mature NGFβ-negative (filled arrowheads) neurons is increased. C: ProNGF-ir (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowhead). D: Following sympathectomy, neurons unstained for ProNGF (filled arrowheads) are increased within cardiac ganglia. E: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in D is 40μm.

    Journal: Autonomic neuroscience : basic & clinical

    Article Title: Modulation of rat parasympathetic cardiac ganglion phenotype and NGF synthesis by adrenergic nerves

    doi: 10.1016/j.autneu.2008.10.012

    Figure Lengend Snippet: NGF protein expression in cardiac ganglia from sham-operated (A, C) and stellate ganglionectomized (B, D) rats. A: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of large control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. B: After sympathectomy, although mature NGFβ-ir (white arrowhead) neurons are observed, the frequency of mature NGFβ-negative (filled arrowheads) neurons is increased. C: ProNGF-ir (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowhead). D: Following sympathectomy, neurons unstained for ProNGF (filled arrowheads) are increased within cardiac ganglia. E: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in D is 40μm.

    Article Snippet: Specificity of the mature NGF and proNGF antibodies was determined by probing immunoblots loaded with 500 ng human recombinant NGF (Sigma); the mature NGFβ antibody primarily detected the 13.5 and 16 kDa mature forms of NGF whereas the proNGF antibody detected bands at 35 and 53 kDa.

    Techniques: Expressing, Staining

    Nerve growth factor (NGF) transcript and protein expression in cardiac ganglia from control (CON; A, C, E, G) and guanethidine-treated (GUAN; B, D, F, H) rats. A: NGF transcripts are expressed strongly in most large cardiac ganglion neurons (white arrowheads); non-transcript expressing neurons (filled arrowheads) are also observed. B: After guanethidine treatment, non-NGF mRNA-expressing neurons (filled arrowheads) are more prominent than positive neurons (white arrowhead). C: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. D: After guanethidine treatment, mature NGFβ-negative neurons (filled arrowheads) are more frequent than those mature NGFβ-ir (white arrowheads). E: ProNGF-ir (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowheads). F: Following guanethidine treatment, ProNGF-ir neurons (white arrowheads), or unstained (filled arrowheads), are observed in similar numbers to controls. G, H: Vesicular acetylcholine transporter (VAChT)-ir is observed in neurons (white arrowheads) from adjacent sections to those processed for mature NGFβ (C, D) and proNGF (E, F). VAChT-negative neurons are indicated by filled arrowheads. I: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in H is 40μm for panels A-H.

    Journal: Autonomic neuroscience : basic & clinical

    Article Title: Modulation of rat parasympathetic cardiac ganglion phenotype and NGF synthesis by adrenergic nerves

    doi: 10.1016/j.autneu.2008.10.012

    Figure Lengend Snippet: Nerve growth factor (NGF) transcript and protein expression in cardiac ganglia from control (CON; A, C, E, G) and guanethidine-treated (GUAN; B, D, F, H) rats. A: NGF transcripts are expressed strongly in most large cardiac ganglion neurons (white arrowheads); non-transcript expressing neurons (filled arrowheads) are also observed. B: After guanethidine treatment, non-NGF mRNA-expressing neurons (filled arrowheads) are more prominent than positive neurons (white arrowhead). C: Mature NGFβ immunoreactivity (ir) is present within a subpopulation of control cardiac ganglion neurons (white arrowheads); many neurons (filled arrowhead) do not express mature NGFβ-ir. D: After guanethidine treatment, mature NGFβ-negative neurons (filled arrowheads) are more frequent than those mature NGFβ-ir (white arrowheads). E: ProNGF-ir (white arrowheads) is prominent in cardiac ganglion neurons; a similar proportion does not stain for ProNGF (filled arrowheads). F: Following guanethidine treatment, ProNGF-ir neurons (white arrowheads), or unstained (filled arrowheads), are observed in similar numbers to controls. G, H: Vesicular acetylcholine transporter (VAChT)-ir is observed in neurons (white arrowheads) from adjacent sections to those processed for mature NGFβ (C, D) and proNGF (E, F). VAChT-negative neurons are indicated by filled arrowheads. I: Quantitative representation of the percentage of large cardiac ganglion neurons immunoreactive for mature NGFβ and proNGF. Scale bar in H is 40μm for panels A-H.

    Article Snippet: Specificity of the mature NGF and proNGF antibodies was determined by probing immunoblots loaded with 500 ng human recombinant NGF (Sigma); the mature NGFβ antibody primarily detected the 13.5 and 16 kDa mature forms of NGF whereas the proNGF antibody detected bands at 35 and 53 kDa.

    Techniques: Expressing, Staining

    PDR-aqueous humor stimulates tube-like structures in a proNGF-dependent manner. HRE cells were grown into confluence then trypsinized and mixed with reduced-growth factor Matrigel and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL). Representative micrographs for alignment of HRE into tube-like structures are shown after 18 hrs of incubation. Statistical analysis of tube length showed that aqueous humor increased mean tube formation 1.7-fold compared to the control group. Addition of anti-proNGF antibodies to aqueous humor samples significantly reduced the relative mean tube length but did not affect control group. Prior treatment of humor samples with rabbit IgG did not significantly reduce relative mean length when compared to the untreated aq. humor group ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Journal: Journal of Diabetes Research

    Article Title: Pronerve Growth Factor Induces Angiogenesis via Activation of TrkA: Possible Role in Proliferative Diabetic Retinopathy

    doi: 10.1155/2013/432659

    Figure Lengend Snippet: PDR-aqueous humor stimulates tube-like structures in a proNGF-dependent manner. HRE cells were grown into confluence then trypsinized and mixed with reduced-growth factor Matrigel and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL). Representative micrographs for alignment of HRE into tube-like structures are shown after 18 hrs of incubation. Statistical analysis of tube length showed that aqueous humor increased mean tube formation 1.7-fold compared to the control group. Addition of anti-proNGF antibodies to aqueous humor samples significantly reduced the relative mean tube length but did not affect control group. Prior treatment of humor samples with rabbit IgG did not significantly reduce relative mean length when compared to the untreated aq. humor group ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Article Snippet: Mutant (cleavage-resistant) proNGF protein and anti-proNGF antibody were purchased from Alomone Labs (Jerusalem, Israel) and IgG was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Incubation

    ProNGF and aqueous humor from PDR patients stimulated cell proliferation. HRE cells were grown into confluence then trypsinized and plated as described in method section. Cells were switched to serum free medium and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL) for 24 hours then cells were trypsinized and counted. Statistical analysis showed that aqueous humor from PDR patients stimulated cell proliferation by 1.8-fold compared to the control group. Adding anti-proNGF antibody to aqueous humor samples significantly reduced the relative number of proliferating cells while IgG did not. Addition of anti-proNGF antibody to HRE cells did not affect number of proliferating cells ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Journal: Journal of Diabetes Research

    Article Title: Pronerve Growth Factor Induces Angiogenesis via Activation of TrkA: Possible Role in Proliferative Diabetic Retinopathy

    doi: 10.1155/2013/432659

    Figure Lengend Snippet: ProNGF and aqueous humor from PDR patients stimulated cell proliferation. HRE cells were grown into confluence then trypsinized and plated as described in method section. Cells were switched to serum free medium and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL) for 24 hours then cells were trypsinized and counted. Statistical analysis showed that aqueous humor from PDR patients stimulated cell proliferation by 1.8-fold compared to the control group. Adding anti-proNGF antibody to aqueous humor samples significantly reduced the relative number of proliferating cells while IgG did not. Addition of anti-proNGF antibody to HRE cells did not affect number of proliferating cells ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Article Snippet: Mutant (cleavage-resistant) proNGF protein and anti-proNGF antibody were purchased from Alomone Labs (Jerusalem, Israel) and IgG was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques:

    PDR-aqueous humor stimulates cell migration in a proNGF-dependent manner. HRE cells were grown to confluence and then scratched using a standard cell scrapper. Cells were switched to serum free medium and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL). Representative micrographs for wounded HME cells are shown after 18 hours of various treatments. Statistical analysis showed that aqueous humor increased mean cell migration by 1.8-fold compared to the control group. Addition of anti-proNGF antibody to aqueous humor samples significantly reduced the mean percent of cell migration to the level of the control group whereas IgG did not significantly impact stimulatory effect of aqueous humor samples. Addition of anti-ProNGF antibody to control HRE cells did not significantly affect percent cell migration compared to control group ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Journal: Journal of Diabetes Research

    Article Title: Pronerve Growth Factor Induces Angiogenesis via Activation of TrkA: Possible Role in Proliferative Diabetic Retinopathy

    doi: 10.1155/2013/432659

    Figure Lengend Snippet: PDR-aqueous humor stimulates cell migration in a proNGF-dependent manner. HRE cells were grown to confluence and then scratched using a standard cell scrapper. Cells were switched to serum free medium and treated with aqueous humor samples (10 μ L/mL) in the presence or absence of either anti-proNGF antibody or rabbit IgG (1 μ g/mL). Representative micrographs for wounded HME cells are shown after 18 hours of various treatments. Statistical analysis showed that aqueous humor increased mean cell migration by 1.8-fold compared to the control group. Addition of anti-proNGF antibody to aqueous humor samples significantly reduced the mean percent of cell migration to the level of the control group whereas IgG did not significantly impact stimulatory effect of aqueous humor samples. Addition of anti-ProNGF antibody to control HRE cells did not significantly affect percent cell migration compared to control group ( ∗,‡ statistically significant compared to control and aqueous humor groups, resp., ( P

    Article Snippet: Mutant (cleavage-resistant) proNGF protein and anti-proNGF antibody were purchased from Alomone Labs (Jerusalem, Israel) and IgG was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Migration

    Immunostaining of NGF-β, p75NTR and trkA in uteri of ICR mice with adenomyosis or not . A: Uterus of 140 ± 5 d control ICR mice stained for NGF-β. B-E: Immnostaining of NGF-β in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. F: Uterus of 140 ± 5 d control ICR mice stained for p75NTR. G-J: Immnostaining of p75NTR in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. K-L: trkA immnoreactivity was detected in nerve fibers (K; arrows, nerve fibers), luminal and glandular epithelial cells of endometrium (L) in uterus of adenomyosis ICR mice aged 140 ± 5 d. (Bar = 100 μm).

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

    doi: 10.1186/1477-7827-9-30

    Figure Lengend Snippet: Immunostaining of NGF-β, p75NTR and trkA in uteri of ICR mice with adenomyosis or not . A: Uterus of 140 ± 5 d control ICR mice stained for NGF-β. B-E: Immnostaining of NGF-β in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. F: Uterus of 140 ± 5 d control ICR mice stained for p75NTR. G-J: Immnostaining of p75NTR in uterus of 90 ± 5 d, 140 ± 5 d, 190 ± 5 d and 240 ± 5 d ICR mice with adenomyosis, respectively. K-L: trkA immnoreactivity was detected in nerve fibers (K; arrows, nerve fibers), luminal and glandular epithelial cells of endometrium (L) in uterus of adenomyosis ICR mice aged 140 ± 5 d. (Bar = 100 μm).

    Article Snippet: Western blot analysis of NGF-β, proNGF and its receptors in the uterus The protein levels of NGF (13 kDa) and its precursors (proNGF, 27 kDa), p75NTR (75 kDa) and trkA (145 kDa), in the uteri of adenomyosis and control mice were detected by western bolts (Figure ).

    Techniques: Immunostaining, Mouse Assay, Staining

    Western-blot analysis of NGF-β, pro NGF, p75NTR and trkA in adenomyosis (Ad) and control (Co) ICR mice uteri . A: Representative Western-blot analysis of NGF-β, pro NGF, p75NTR and trkA in adenmoysis and control mice in different age groups. B: Densitometirc analysis of NGF-β, pro NGF, p75NTR and trkA in adenomyosis and control mice in different age groups expressed as a percentage after actin, beta normalization (n = 5; a = 90 ± 5 d, b = 150 ± 5 d, c = 190 ± 5 d, d = 240 ± 5d; *P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

    doi: 10.1186/1477-7827-9-30

    Figure Lengend Snippet: Western-blot analysis of NGF-β, pro NGF, p75NTR and trkA in adenomyosis (Ad) and control (Co) ICR mice uteri . A: Representative Western-blot analysis of NGF-β, pro NGF, p75NTR and trkA in adenmoysis and control mice in different age groups. B: Densitometirc analysis of NGF-β, pro NGF, p75NTR and trkA in adenomyosis and control mice in different age groups expressed as a percentage after actin, beta normalization (n = 5; a = 90 ± 5 d, b = 150 ± 5 d, c = 190 ± 5 d, d = 240 ± 5d; *P

    Article Snippet: Western blot analysis of NGF-β, proNGF and its receptors in the uterus The protein levels of NGF (13 kDa) and its precursors (proNGF, 27 kDa), p75NTR (75 kDa) and trkA (145 kDa), in the uteri of adenomyosis and control mice were detected by western bolts (Figure ).

    Techniques: Western Blot, Mouse Assay

    mRNA expression of Ntrk1 and Ngfr in DRGs of adenomyosis (Ad) and control (Co) mice . A: the mRNA level of Ntrk1 in DRGs of adenomyosis and control mice for different age groups. B: the Ngfr level in DRGs of adenomyosis and control mice for different age groups. (n = 4; *P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

    doi: 10.1186/1477-7827-9-30

    Figure Lengend Snippet: mRNA expression of Ntrk1 and Ngfr in DRGs of adenomyosis (Ad) and control (Co) mice . A: the mRNA level of Ntrk1 in DRGs of adenomyosis and control mice for different age groups. B: the Ngfr level in DRGs of adenomyosis and control mice for different age groups. (n = 4; *P

    Article Snippet: Western blot analysis of NGF-β, proNGF and its receptors in the uterus The protein levels of NGF (13 kDa) and its precursors (proNGF, 27 kDa), p75NTR (75 kDa) and trkA (145 kDa), in the uteri of adenomyosis and control mice were detected by western bolts (Figure ).

    Techniques: Expressing, Mouse Assay

    Characterization of proNGF and GFP expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

    doi: 10.1016/j.bbadis.2017.12.023

    Figure Lengend Snippet: Characterization of proNGF and GFP expression within retinal vasculature. (a) Representative images from WT, Cre and Cre-proNGF frozen retinal sections showing vasculature stained with isolectin B-4 (red) and anti-proNGF (green). Note the colocalization (yellow) of proNGF is prominent in the superficial capillaries at the ganglion cell layer (GCL) and deep retina vessels at the inner plexiform layer (IPL) of Cre-proNGF group, but not other groups. (b) Representative images from WT, Cre and Cre-proNGF showing vasculature stained with isolectin B-4 (red) and anti-GFP-protein (green). Note co-localization (yellow) of GFP staining within retinal capillaries in Cre-proNGF group, but not in WT or Cre-control.

    Article Snippet: After 4-weeks of proNGF expression, frozen retinal cryo-sections (10-μm thickness) were permealized, blocked in goat serum and incubated with primary antibodies including anti-GFP (Abcam, Rabbit polyclonal, 1:200), anti-proNGF (Alomone, Israel, Rabbit polyclonal, 1:100), anti-GFAP (polyclonal, Affinity Bioreagents, Rockford, IL, USA; 1:200), followed by Oregon-green or Texas-red conjugated goat antirabbit secondary antibody (Invitrogen, Carlsbad, CA, USA; 1:500).

    Techniques: Expressing, Staining

    Double immunofluorescence staining revealed that proNGF + cells (FITC, green) were reactive for NeuN (CY3, red) (A~D), GFAP (CY3, E~H) and OX42 (CY3, I~L). Scale bars in A~D, I~L=50μm, E~H=100μm.

    Journal: Journal of the neurological sciences

    Article Title: Bone Marrow Stromal Cell Therapy Reduces proNGF and p75 Expression in Mice with Experimental Autoimmune Encephalomyelitis

    doi: 10.1016/j.jns.2008.12.033

    Figure Lengend Snippet: Double immunofluorescence staining revealed that proNGF + cells (FITC, green) were reactive for NeuN (CY3, red) (A~D), GFAP (CY3, E~H) and OX42 (CY3, I~L). Scale bars in A~D, I~L=50μm, E~H=100μm.

    Article Snippet: In addition, the following antibodies were used to measure the cell sources of proNGF and p75: NeuN (1:500, Chemicon, MAB377) for neurons; glial fibrillary acidic protein (GFAP, 1:10000, DAKO, Z0334) for astrocytes; anti-CNPase (1:100, Chemicon, MAB326R) for oligodendrocytes; OX42 (1:300, Accurate Chemical, MCA275R) for microglia cells.

    Techniques: Double Immunofluorescence Staining