Journal: PLoS ONE

Article Title: Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

doi: 10.1371/journal.pone.0047316

Figure Lengend Snippet: Confluent myoFB were exposed to NGF at the indicated doses/times and evaluated for biochemical and molecular changes. (A) αSMA protein expression in both myoFB and FB, as detected by cell surface ELISA (optic density) and flow cytometry (fluorescence intensity). (B–C) Fluorescent histograms showing a different pattern of trkA NGFR and p75 NTR expression in acute and chronic 100 ng/mL NGF exposed myoFB, as compared to untreated myoFB (black line). Mean Fluorescent Intensity (MFI) plots are shown inside fluorescent histograms (iso stands for isotype-matched control FI; 7.05 for trkA NGFR and 8.39 for p75 NTR ). (D) Morphological distribution of trkA NGFR /p75 NTR in untreated, acute and chronic 100 ng/mL NGF treated myoFB. (E–F) Target gene expression specific for trkA NGFR /p75 NTR after acute and for αSMA/p75 NTR after chronic exposure to increasing NGF (p<.05).

Article Snippet: Purified 2.5S-NGF (GradeI) and purified human TGFβ1 were provided by Alomone Labs Ltd. (Jerusalem, Israel) and PeproTech EC Ltd (London, UK) respectively; neutralizing anti-pan TGFβ antibodies and neutralizing anti-NGF antibodies (500 ng/mL) were from R&D Systems, Minneapolis, MN; neutralizing anti-trkA NGFR (100 ng/mL) and anti-p75 NTR (100 ng/mL) were from Calbiochem Novabiochem Corp. (San Diego, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

doi: 10.1371/journal.pone.0047316

Figure Lengend Snippet: (A) Pictures depicting TUNEL reactivity in p75 NTR positive cells exposed to chronic 100 ng/mL NGF, as compared to TGFβ1 exposed cells. Changes in morphology, such as having rounded cytoplasm and markedly condensed chromatin/nuclear fragmentation, are clear visible in p75 NTR -bearing myoFB. A few TUNEL-positive myoFB were also quantified in untreated cultures (not shown). (B) Overlays and single staining specific for p75 NTR and cleaved caspase3 are visible in both acute and chronic 100 ng/mL NGF exposed myoFB, as compared to untreated myoFB. (C–D) p75 NTR positive sorted cells analysed for active caspase3 expression and related statistical analysis of MFI. Note the specific reduction upon pretreated with trkA NGFR and/or p75 NTR chemical inhibitors.

Article Snippet: Purified 2.5S-NGF (GradeI) and purified human TGFβ1 were provided by Alomone Labs Ltd. (Jerusalem, Israel) and PeproTech EC Ltd (London, UK) respectively; neutralizing anti-pan TGFβ antibodies and neutralizing anti-NGF antibodies (500 ng/mL) were from R&D Systems, Minneapolis, MN; neutralizing anti-trkA NGFR (100 ng/mL) and anti-p75 NTR (100 ng/mL) were from Calbiochem Novabiochem Corp. (San Diego, USA).

Techniques: TUNEL Assay, Staining, Expressing

Journal: PLoS ONE

Article Title: Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

doi: 10.1371/journal.pone.0047316

Figure Lengend Snippet: (A) A flow chart schematizing the siRNA experiments. (B) Confocal images depicting GFP positive cells (image) and p75 NTR -GFP double-positive cells (insert over the image), re-plated 5 hrs after electroporation. (C–D) Representative histogram for GFP specific staining, in comparison to control siRNA (control cells), showing a decreased p75 NTR positive expression and the related p75 NTR target gene deregulation. (E) Mean Fluorescence Intensity (MFI) specific for caspACE-VAD comparing 100 ng/mL NGF and 10 ng/mL TGFβ1 effects on control-siRNA, p75 NTR siRNA and double trkA NGFR /p75 NTR siRNA treated cells. A 37.5%-decrease (*) and a 50%-decrease (**) of apoptotic signal were detected respectively in p75 NTR -siRNA and p75 NTR /trkA NGFR -siRNA (p<.05).

Article Snippet: Purified 2.5S-NGF (GradeI) and purified human TGFβ1 were provided by Alomone Labs Ltd. (Jerusalem, Israel) and PeproTech EC Ltd (London, UK) respectively; neutralizing anti-pan TGFβ antibodies and neutralizing anti-NGF antibodies (500 ng/mL) were from R&D Systems, Minneapolis, MN; neutralizing anti-trkA NGFR (100 ng/mL) and anti-p75 NTR (100 ng/mL) were from Calbiochem Novabiochem Corp. (San Diego, USA).

Techniques: Electroporation, Staining, Expressing, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: Effect of intracerebroventricular infusion of NGF on the loss and recovery of the cholinergic phenotype . A , The septohippocampal path was axotomized and NGF-infusion was performed for two weeks. Left, quantification of septal cholinergic neurons in the lesioned side as compared to the contralateral side in untreated animals (control), lesioned animals (lesion) and lesioned animals brain-infused with NGF (lesion + NGF sim.). Right, light microscopy of anti-NGF immunohistochemistry showing the wide distribution of infused NGF and uptake of NGF by septal neurons. Exogenous NGF infused right after axotomy protects cholinergic cells from ChAT-loss. B , The septohippocampal path was axotomized and 3 weeks after the axotomy (delayed NGF-infusion), NGF was infused for two additional weeks. This procedure did not protect cholinergic cells from ChAT-loss, contrary to the protection observed with simultaneous infusion. C , Confocal microscopy of double immunofluorescence against TrkA and ChAT showing the colocalization of these markers in septal neurons 21 days after axotomy (inferior panel). There are no ChAT-negative cells positive for TrkA, suggesting that neurons cannot respond to NGF 3 weeks post-axotomy due to down-regulation of NGF receptors.

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Light Microscopy, Immunohistochemistry, Confocal Microscopy, Immunofluorescence

Journal: Journal of Aging Research

Article Title: Cholinergic Degeneration and Alterations in the TrkA and p75NTR Balance as a Result of Pro-NGF Injection into Aged Rats

doi: 10.4061/2011/460543

Figure Lengend Snippet: Intra-hippocampal injections of pro-NGF significantly increased hippocampal expression of pro-NGF (a), but did not alter basal forebrain pro-NGF levels (b). The pro-NGF antibody was specific for pro-NGF (~32 kD) and did not detect mature NGF protein levels as shown in a pro-NGF treated animal; when incubated with pro-NGF blocking peptide, no bands were present (c). Changes in protein levels were normalized to β -Actin; * P ≤ .05.

Article Snippet: Primary antibodies used include rabbit anti-TrkA (1 : 1000, kindly provided by Dr. Louis Reichardt), mouse anti- β -Actin (1 : 10,000, Sigma, St. Louis, MO), mouse anti-p75NTR (1 : 1000, Millipore, Bellerica, MA), rabbit anti-caspase-3 (1 : 1,000, Stressgen, Ann Arbor, MI), rabbit antisortilin (1 : 1000, Abcam, Cambridge, MA), rabbit antiplasminogen (1 : 500, R&D Systems Inc., Minneapolis, MN), rabbit anti-Pro : NGF (1 : 200, Alomone Labs, Jerusalem, Israel) and secondary antibodies include donkey anti-rabbit HRP (1 : 5000, Jackson Immunoresearch, West Grove, PA), donkey antimouse HRP (1 : 5000, Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Incubation, Blocking Assay