prongf  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: Expression of NGF/proNGF and Their Receptors TrkA, p75NTR and Sortilin in Melanoma

doi: 10.3390/ijms23084260

Figure Lengend Snippet: proNGF expression in nevi, primary melanoma and metastatic human tissues ( A – F ). Immunohistochemical detection of proNGF, representative pictures are shown for compound nevi ( A ), dysplastic nevi ( B ), thin primary melanomas ( C ), thick primary melanomas ( D ), lymph node metastases ( E ) and distant metastases ( F ). ( G ) Digital quantification of proNGF staining intensities according to grouped pathological subtypes: nevi (h-score = 156.2, IQR 138.9–176.1), primary melanomas (h-score = 129.0, IQR 111.8–1148.1) and metastases (h-score = 115.1, IQR 93.33–130.1). ( H ) proNGF staining intensities for individual pathological subtypes: CN = compound nevi (h-score = 143.4, IQR 126.1–176.0), DN = dysplastic nevi (h-score = 158.6, IQR 143.0–189.6), TnP = thin primary (h-score = 125.3, IQR 111.8–134.6), TkP = thick primary (h-score = 133.6, IQR 109.2–154.1), LNM = lymph node metastasis (h-score = 116.7, IQR 105.9–132.3), DM = distant metastasis (h-score = 101.8, IQR 91.64–129.7). ( I ) Correlation of proNGF and NGF stain intensities. Scale bar = 90 µm. Data are expressed as medians (horizontal line in the center of the box) and box limits indicate the interquartile range (IQR) with the whiskers extending 1.5 times the IQR from the 25th and 75th percentiles; outliers are represented by dots. H-score distributions were compared using the Wilcoxon rank-sum (dichotomous) or Kruskal–Wallis (multiple comparisons) tests ( p

Article Snippet: The following primary antibodies were applied: anti-proNGF (0.8 µg/mL, catalogue number ANT005, Alomone labs, Jerusalem, Israel), anti-NGF (13.3 µg/mL, catalogue number ab52918, Abcam, VIC, Australia), anti-sortilin (0.8 µg/mL, catalogue number ANT009, Alomone labs, Jerusalem, Israel), anti-p75NTR (2 µg/mL, catalogue number ANT007, Alomone labs, Jerusalem, Israel), anti-TrkA (1:200 dilution, catalogue number cs-2508, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Immunohistochemistry, Staining

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

Journal: Cell Death & Disease

Article Title: Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells

doi: 10.1038/s41419-019-1758-z

Figure Lengend Snippet: Effect of p75NTR stimulation on lipid-associated genes in Huh7 cells. Huh7 cells were stimulated with 100 ng/ml NGF or 20 ng/ml pro-NGF for 48 h, and the expression of lipid-associated genes was analyzed by qPCR. Primers are shown in Table 2 . Results were normalized to GAPDH and controls set to 1. Values are mean ± SD, n = 3-4. *** p

Article Snippet: The filters were incubated for 1 h in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 0.1% Tween 20, and 5% skimmed milk or 5% BSA at room temperature and then overnight at 4 °C with primary antibodies as follows: anti-NGF (1:1000; Alomone Labs, AN-240), anti-Pro-NGF (1:500; Alomone labs, ANT-005), anti-p75NTR intracellular region (1:5000; Millipore, 07-476), anti-p75NTR (1:1000; Cell Signaling, 8238T), anti-Caspase-2 (1:1000; Enzo Life Sciences, 11B4), anti-phospho-Caspase2 (1:200; Eurogentec), anti-Caspase 3 (1:1000; Cell Signaling, 9665), anti-cleaved-Caspase 3 (1:1000; Cell Signaling, 9664), anti-SREBP2 Carboxyl terminal region (1:500; BD Biosciences, 557037), anti-SREBP1 (1:1000; Novus Biologicals, NB600-582), anti-Flag (1:2000; Sigma, F1804), and anti-β-actin (1:10,000; Sigma).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells

doi: 10.1038/s41419-019-1758-z

Figure Lengend Snippet: Schematic view of the role of caspase-2 in p75NTR signaling and lipid metabolism. We propose the following scheme for the action of p75NTR and of caspase-2 in SREBP2 and associated gene regulation. Stimulation of p75NTR by NGF or pro-NGF activates the signaling protein p38 MAPK that can phosphorylate caspase-2 (CASP2) leading to the release of caspase-3 (CASP3) from its complex. CASP3, in turn, can cleave SREBP molecules to yield an active transcription factor. SREBP responsive genes include LDLR and other lipogenic genes but also CASP2 itself. Normally SREBP is processed by the enzymes S1P and S2P via a feedback inhibition determined by the cell cholesterol level. We hypothesize that the steroid-mediated and neurotrophin/p75NTR-induced SREBP pathways are distinct but functionally linked

Article Snippet: The filters were incubated for 1 h in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 0.1% Tween 20, and 5% skimmed milk or 5% BSA at room temperature and then overnight at 4 °C with primary antibodies as follows: anti-NGF (1:1000; Alomone Labs, AN-240), anti-Pro-NGF (1:500; Alomone labs, ANT-005), anti-p75NTR intracellular region (1:5000; Millipore, 07-476), anti-p75NTR (1:1000; Cell Signaling, 8238T), anti-Caspase-2 (1:1000; Enzo Life Sciences, 11B4), anti-phospho-Caspase2 (1:200; Eurogentec), anti-Caspase 3 (1:1000; Cell Signaling, 9665), anti-cleaved-Caspase 3 (1:1000; Cell Signaling, 9664), anti-SREBP2 Carboxyl terminal region (1:500; BD Biosciences, 557037), anti-SREBP1 (1:1000; Novus Biologicals, NB600-582), anti-Flag (1:2000; Sigma, F1804), and anti-β-actin (1:10,000; Sigma).

Techniques: Inhibition

Journal: Cell Death & Disease

Article Title: Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells

doi: 10.1038/s41419-019-1758-z

Figure Lengend Snippet: NGF is expressed in fatty liver of obese mice together with p75NTR. a NGF has increased in leptin-deficient ob/ob mouse livers compared with control, wild-type (wt) mice. b p75NTR was expressed in liver with no significant change between ob/ob and control mice. c , d The level of NGF has increased in leptin-receptor-deficient db/db mouse liver in comparison to controls, c immunoblot analysis and d quantification. ** p

Article Snippet: The filters were incubated for 1 h in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 0.1% Tween 20, and 5% skimmed milk or 5% BSA at room temperature and then overnight at 4 °C with primary antibodies as follows: anti-NGF (1:1000; Alomone Labs, AN-240), anti-Pro-NGF (1:500; Alomone labs, ANT-005), anti-p75NTR intracellular region (1:5000; Millipore, 07-476), anti-p75NTR (1:1000; Cell Signaling, 8238T), anti-Caspase-2 (1:1000; Enzo Life Sciences, 11B4), anti-phospho-Caspase2 (1:200; Eurogentec), anti-Caspase 3 (1:1000; Cell Signaling, 9665), anti-cleaved-Caspase 3 (1:1000; Cell Signaling, 9664), anti-SREBP2 Carboxyl terminal region (1:500; BD Biosciences, 557037), anti-SREBP1 (1:1000; Novus Biologicals, NB600-582), anti-Flag (1:2000; Sigma, F1804), and anti-β-actin (1:10,000; Sigma).

Techniques: Mouse Assay

Journal: Cell Death & Disease

Article Title: Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells

doi: 10.1038/s41419-019-1758-z

Figure Lengend Snippet: p75NTR stimulation activated SREBP1 and SREBP2 in Huh7 hepatocyte cells. Human Huh7 cells were stimulated with 10 ng/ml pro-NGF or 50 ng/ml NGF for 16 h followed by immunoblotting using antibodies to detect the presence of cleaved/activated SREBP1 and SREBP2. a SREBP1, immunoblots and b quantification. Values are mean ± SD, n = 3. ** P

Article Snippet: The filters were incubated for 1 h in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 0.1% Tween 20, and 5% skimmed milk or 5% BSA at room temperature and then overnight at 4 °C with primary antibodies as follows: anti-NGF (1:1000; Alomone Labs, AN-240), anti-Pro-NGF (1:500; Alomone labs, ANT-005), anti-p75NTR intracellular region (1:5000; Millipore, 07-476), anti-p75NTR (1:1000; Cell Signaling, 8238T), anti-Caspase-2 (1:1000; Enzo Life Sciences, 11B4), anti-phospho-Caspase2 (1:200; Eurogentec), anti-Caspase 3 (1:1000; Cell Signaling, 9665), anti-cleaved-Caspase 3 (1:1000; Cell Signaling, 9664), anti-SREBP2 Carboxyl terminal region (1:500; BD Biosciences, 557037), anti-SREBP1 (1:1000; Novus Biologicals, NB600-582), anti-Flag (1:2000; Sigma, F1804), and anti-β-actin (1:10,000; Sigma).

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells

doi: 10.1038/s41419-019-1758-z

Figure Lengend Snippet: Role of Caspase-2 in p75NTR mediated regulation of SREBP. a Structure of human caspase-2 (CASP2) showing the CARD domain and the 18 and 13 kDa caspase subunits. p38 MAPK phosphorylation site at threonine (Thr)180 in CASP2 is conserved among species. The amino acid numbering underlying functional domains in CASP2 is shown. Boxes represent sequence homology among mammalian species. b Phosphoprotein retardation was performed as described in Materials and methods. A specific phospho-CASP2 antibody against Thr180 was used in conjunction with an antibody against CASP2, recognizing non-phosphorylated form of the enzyme. Phosphorylated CASP2 migrated slower in the Phos-Tag gel (right) as compared with the protein run on denaturating SDS-PAGE (left). c , d Huh7 hepatocyte cells were stimulated with 50 ng NGF ( c ) or 10 ng/ml pro-NGF ( d ) for different times followed by immunoblotting using phospho-CASP2 and anti-CASP2 antibodies. β-Actin was used as a control. e , f Cells were treated with pro-NGF for 6 h in the absence or presence 1 μM of the p38 MAPK inhibitor, SB203580 (SB). e Immunoblot and f quantification of p-CASP2 levels. SB reduced the increase in p-CASP2 by pro-NGF. Values are means ± SD, n = 4. * p

Article Snippet: The filters were incubated for 1 h in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 0.1% Tween 20, and 5% skimmed milk or 5% BSA at room temperature and then overnight at 4 °C with primary antibodies as follows: anti-NGF (1:1000; Alomone Labs, AN-240), anti-Pro-NGF (1:500; Alomone labs, ANT-005), anti-p75NTR intracellular region (1:5000; Millipore, 07-476), anti-p75NTR (1:1000; Cell Signaling, 8238T), anti-Caspase-2 (1:1000; Enzo Life Sciences, 11B4), anti-phospho-Caspase2 (1:200; Eurogentec), anti-Caspase 3 (1:1000; Cell Signaling, 9665), anti-cleaved-Caspase 3 (1:1000; Cell Signaling, 9664), anti-SREBP2 Carboxyl terminal region (1:500; BD Biosciences, 557037), anti-SREBP1 (1:1000; Novus Biologicals, NB600-582), anti-Flag (1:2000; Sigma, F1804), and anti-β-actin (1:10,000; Sigma).

Techniques: Functional Assay, Sequencing, SDS Page

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

Techniques: SPR Assay

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

Techniques: Incubation

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

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

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

Techniques: Binding Assay, Chromatin Immunoprecipitation, SPR Assay

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: A representative WB, challenged both with anti-proNGF and anti-NGF, is shown in Figure .

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