Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: Silencing p75 NTR prevents proNGF accumulation and restores NGF levels in rat retina. Rats received single intraocular injection of one of the following: pGFP plus scrambled shRNA (GFP+Scr), pGFP plus shRNA against p75 NTR (GFP+shRNA); pGFP-proNGF123, a cleavage-resistant form of proNGF plus scrambled shRNA (proNGF+Scr); pGFP-proNGF123 plus shRNA against p75 NTR (proNGF+shRNA). Retinas were lysed after 6 weeks. ( a ) Representative western blot image and ( b,c ) statistical analysis of total p75 NTR (75 kDa) and its intracellular domain (ICD, 25 kDa) normalized to actin. A 2 × 2 analysis showed a significant interaction between proNGF overexpression and silencing p75 NTR . Overexpression of proNGF induced expression of both total p75 NTR and p75 ICD compared with GFP. Silencing p75 NTR with shRNA prevented the increase in p75 NTR and p75 ICD expression in proNGF group but not in GFP group. n = 4–5 per group, * P < 0.05 versus other groups. ( d ) Representative western blot image and statistical analysis of proNGF expression normalized to actin showing significant increases in proNGF in proNGF overexpressing group that were mitigated by silencing p75 NTR compared with GFP controls. ( e ) Representative western blot images and statistical analysis of NGF expression normalized to actin. A 2 × 2 analysis showed a significant interaction between proNGF overexpression and silencing p75 NTR . ProNGF overexpression resulted in significant decreases in NGF levels compared with GFP. Silencing p75 NTR with shRNA significantly reduced NGF levels in GFP-controls and restored NGF levels in proNGF overexpressing retina. Results presented as mean ± SD. n = 4–5 per group. * P < 0.05 versus other groups.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Injection, shRNA, Western Blot, Over Expression, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: Silencing p75 NTR suppresses proNGF-induced vascular cell death and apoptotic marker in rat retina. Representative images of trypsin-digested retinas stained with TUNEL (green) and counterstained with DAPI (blue) are shown for ( a ) GFP+Scr, ( b ) GFP+p75shRNA, ( c ) proNGF+Scr, and ( d ) proNGF+p75shRNA. TUNEL assay showed several positive cells in proNGF-overexpressing retinas, suggesting that capillary degradation occurs through apoptosis. ( e ) Representative western blots and statistical analysis of pJNK and JNK and ( f ) cleaved-PARP normalized to actin. 2 × 2 analysis showed interaction between proNGF expression and silencing p75 NTR . Overexpression of proNGF induced significant activation of JNK and cleavage of PARP compared with GFP-controls. These effects were mitigated by p75shRNA but not scrambled. Results presented as mean ± SD. n = 4–6 per group. * P < 0.05 versus other groups.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Marker, Staining, TUNEL Assay, Western Blot, Expressing, Over Expression, Activation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: Expression of proNGF is dependent on p75 NTR in EC. Human retinal ECs were transduced with one of the followings: pGFP plus scrambled siRNA (GFP+Scr), pGFP plus siRNA against p75 NTR (GFP+siRNA); pGFP-proNGF123, a mutant cleavage resistant form of proNGF, plus scrambled siRNA (proNGF+Scr); pGFP-proNGF123 plus siRNA against p75 NTR (proNGF+siRNA), and cell lysate and supernatant were collected after 48 hours. ( a,b ) Representative western blots statistical analysis of total p75 NTR expression normalized to actin. ( a,c ) Representative western blots and statistical analysis of proNGF expression normalized to actin. 2 × 2 analysis showed interaction between proNGF overexpression and silencing p75 NTR . Silencing p75 NTR significantly reduced its expression in both GFP-controls and proNGF-groups. Results showed that overexpression of proNGF induced p75 NTR expression and that silencing p75 NTR downregulated proNGF expression in EC. Results presented as mean ± SD. n = 5–6 per group. * P < 0.05 versus other groups. ( d ) Representative western blots and statistical analysis of NGF expression normalized to actin in EC. ( e ) Representative western blots and statistical analysis of TrkA activation normalized to total TrkA. 2 × 2 analysis demonstrated interaction between proNGF overexpression and silencing p75 NTR . Overexpression of proNGF resulted in significant decreases in NGF levels and activation of its TrkA receptor compared with GFP-controls. Silencing p75 NTR expression increased NGF levels and enhanced TrkA activation in proNGF overexpressing cells. Results are expressed as mean ± SD. n = 5 per group. * P < 0.05 versus other groups.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Expressing, Transduction, Mutagenesis, Western Blot, Over Expression, Activation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: Silencing p75 NTR suppresses proNGF-induced apoptotic markers in EC. Representative western blots and statistical analysis of ( a ) pJNK, total JNK, ( b ) pP38MAPK, total p38MAPK, and ( c ) cleaved-PARP (C-PARP), actin in EC lysates. 2 × 2 way analysis showed interaction between proNGF overexpression and silencing p75 NTR . Overexpression of proNGF induced EC apoptosis evident by activation of JNK, p38MAPK, and cleavage of PARP. These effects were blunted by silencing p75 NTR . Results presented as mean ± SD. n = 4–6 per group. * P < 0.05 versus other groups.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Western Blot, Over Expression, Activation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: ProNGF causes protein interaction and nuclear translocation of p75 NTR and neurotrophin interacting factor (NRIF). Human retinal endothelial cells were treated with human mutant (hm-proNGF, 50 ng/ml) in the presence or absence of a selective p75 NTR antagonist (A, 20 μmol/l) for 48 hours. ( a ) Cell death was assessed by measuring activity of caspase enzyme and the results were presented as mean ± SD. n = 3–5 per group. * P < 0.05 versus other groups. ( b ) Representative images of immunolocalization showing that hm-proNGF induced nuclear translocation of NRIF (green) within the nucleus (blue) that was blocked by p75 NTR antagonist (A) (proNGF+A). ( c ) Enlarged micrographs of EC treated with hm-proNGF showing colocalization of p75 NTR (red), NRIF (green) within the nuclei (blue). Similar results were observed in two independent experiments. ( d ) Representative blots and statistical analysis showing that hmproNGF induced protein association between NRIF (90 kDa) and p75 ICD (17 kDa) in EC lysate immunoprecipitated with anti-p75 NTR and immunoblotted by anti-NRIF. Additional band for NRIF was detected at 100 kDa. Results presented as mean ± SD. n = 4 per group. * P < 0.05 versus all groups.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Translocation Assay, Mutagenesis, Activity Assay, Immunoprecipitation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing p75 NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

doi: 10.1038/mtm.2015.13

Figure Lengend Snippet: Schematic diagram depicting the proposed role of proNGF/p75NTR in mediating EC death and development of acellular capillaries. Overexpression of proNGF impairs endogenous NGF/TrkA survival signaling pathway and activates p75NTR-mediated apoptotic signal. ProNGF triggers interaction of intracellular domain of p75NTR (p75 ICD ) and the transcriptional factor neurotrophin interacting factor (NRIF) to activate JNK apoptotic signal. Targeting p75 NTR prevents the apoptotic signal and stimulates endogenous cell survival.

Article Snippet: Antibodies were purchased as follows: anti-proNGF and anti-NGF (Alomone Labs, Jerusalem, Israel), JNK, p-JNK, p-p38, p38MAPK (Santa Cruz Biotechnology), and cleaved-PARP (Cell Signaling, Danvers, MA).

Techniques: Over Expression

Journal: Scientific Reports

Article Title: Innervation of papillary thyroid cancer and its association with extra-thyroidal invasion

doi: 10.1038/s41598-020-58425-5

Figure Lengend Snippet: Nerve density in thyroid cancers, stratified by tumoural proNGF expression. Box (IQR) and whisker (5–95%) plot of nerve density, stratified by the presence or absence of proNGF expression in the primary tumour. Dark grey boxes compare density of nerves per cm 2 of thyroid lobe containing PTC or FTC (medians 8.5 vs 11.4, p = 0.10). Light grey boxes compare nerve density per cm 2 of PTC or FTC (medians 10.3 vs 14.3 p = 0.07).

Article Snippet: Staining of primary tumours for the precursor for nerve growth factor (proNGF) was performed using the automated Ventana platform and the anti-rabbit polycloncal proNGF antibody (Ab9040, Merck Millipore, Darmstadt, Germany) at a dilution of 1:350.

Techniques: Expressing, Whisker Assay

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 2. Signaling through proNGF–p75NTR, but not mNGF–TrkA, is required for Gb3- induced mechanical sensitization. (A) Experimental timeline of the von Frey tests and the i.pl. injection of Gb3 and anti body/antiserum or antibody/antiserum alone. (B, C) The antiserum (1 μl/paw, i.pl.) containing anti-proNGF antibody effectively recovered the Gb3-induced decrease of the 50% paw withdrawal threshold (PWT; 1 h: H = 9.40, P = 0.009; 3 h: H = 9.13, P = 0.010; 6 h: H = 6.79, P = 0.034; 24 h: H = 6.24, P = 0.044) and its area under the curve (AUC; H = 10.1, P = 0.006). n = 8 per group. The injection volume was 10 μl. (D, E) The anti-NGF antibody (1 μg/paw and 10 μg/ paw, i.pl.) failed to significantly alleviate reductions in the pain threshold and its AUC (H = 10.5, P = 0.015) in Gb3-injected mice. Vehicle, n = 7; Gb3 + Control IgG, n = 8; Gb3 + Anti-NGF antibody (1 μg), n = 9; Gb3 + Anti-NGF antibody (10 μg), n = 6. The injection volume was 10 or 20 μl. (F, G) Administration of an anti-p75NTR antibody (1 μg/paw, i.pl.) almost completely reversed the Gb3-induced decline of the 50% PWT (24 h: H = 8.44, P = 0.015) and its AUC (H = 9.16, P = 0.010) to the level of the vehicle control. Vehicle, n = 11; Gb3 + Control IgG, n = 15; Gb3 + Anti-p75NTR antibody, n = 14. The injection volume was 10 or 20 μl. (H, I) Inhibition of TrkA activity with the neutral izing antibody (1 μg/paw and 10 μg/paw, i. pl.) gave minimal influence on the Gb3- induced mechanical allodynia (AUC: H = 9.80, P = 0.020). Vehicle, n = 11; Gb3 + Control IgG, n = 11; Gb3 + Anti-TrkA anti body (1 μg), n = 7; Gb3 + Anti-TrkA anti body (10 μg), n = 7. The injection volume was 20 μl. *P < 0.05, **P < 0.01 vs the Vehicle group, #P < 0.05, ##P < 0.01 vs the Gb3 + Normal serum or Control IgG group by Dunn’s test with Holm adjustment.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Control, Inhibition, Activity Assay

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 3. Gb3 injection did not lead to elevation of proNGF and mNGF or their receptor expression in either plantar skin or dorsal root ganglia. There were no changes in proNGF (A, C) nor mNGF (B, D) content in the plantar skin (A, B) and dorsal root ganglia (DRG; C, D) in mice that were 1 h or 24 h post-Gb3 treatment. n = 9 per group. The injection volume was 10 or 20 μl. Gb3 injection did not modify the expression levels of p75NTR in the plantar skin (E, 1 h: n = 6 per group; 24 h: n = 7 per group) or the DRG (F, n = 7 per group). The injection volume was 10 μl. The Vehicle (Veh) group, open circles; the Gb3 group, closed circles; MW, molecular weight.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Expressing, Molecular Weight

Journal: BioMed Research International

Article Title: Imbalance of the Nerve Growth Factor and Its Precursor as a Potential Biomarker for Diabetic Retinopathy

doi: 10.1155/2015/571456

Figure Lengend Snippet: Shedding of p75 NTR is consistent in vitreous and serum. (a) Representative bands show p75 NTR expression in vitreous and serum for diabetic compared to nondiabetic control groups. The full length p75 NTR (75 kD) and receptor ectodomain (50 kD) had similar levels of expression in control and diabetic (DB) groups of both vitreous and serum. The possible proteolytic C terminal fragment (CTF) and intracellular domain (ICD) appeared at 27 kD and 22 kD. Differences in expression patterns between vitreous and serum as well as between diabetic and control groups were evident for both CTF and ICD. (b) In vitreous, 27 kD p75 NTR receptor fragment was significantly increased in diabetic (1.65-fold ± 0.23) compared to nondiabetic control group ( N = 4–11, * P < 0.05). (c) In serum, a significant increase in 22 kD p75 NTR occurred in diabetic samples (1.85-fold ± 0.30) compared to nondiabetic controls ( N = 6–10, * P < 0.05).

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-NGF and anti-proNGF (Alomone Labs, Israel) and rabbit polyclonal p75 NTR (1 : 5000), a kind gift from Dr. Bruce Carter, Vanderbilt University, Nashville, TN.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Imbalance of the Nerve Growth Factor and Its Precursor as a Potential Biomarker for Diabetic Retinopathy

doi: 10.1155/2015/571456

Figure Lengend Snippet: Expression of p75 NTR receptor is consistent in vitreous and serum. Results are shown for p75 NTR expression in vitreous and serum of diabetic (DB) and control participants normalized to Ponceau S and respective controls. Full length p75 NTR receptor (75 kD) was not significantly different in diabetic sample compared to control groups in either (a) vitreous ( N = 4–11) or (b) serum ( N = 6–9). The p75 NTR ectodomain (50 kD) was also not significantly different in diabetic compared to control groups in (c) vitreous ( N = 4–11) or (d) serum ( N = 6–9).

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-NGF and anti-proNGF (Alomone Labs, Israel) and rabbit polyclonal p75 NTR (1 : 5000), a kind gift from Dr. Bruce Carter, Vanderbilt University, Nashville, TN.

Techniques: Expressing