Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Increased Nerve Growth Factor Signaling in Sensory Neurons of Early Diabetic Rats Is Corrected by Electroacupuncture

doi: 10.1155/2013/652735

Figure Lengend Snippet: NGF protein and mRNA levels following STZ and EA. Four weeks after STZ injection, an elevation of NGF protein content (a), as measured by ELISA, was found in the lumbar DRGs of adult rats. EA applied for three weeks, starting one week after STZ, restored baseline levels of DRG's NGF in diabetic rats. Data from NGF ELISA are presented as mean ± SD; n = 10 for each experimental group. * P < 0.05 versus control group. # P < 0.05 versus STZ group. NGF mRNA was measured by semiquantitative real-time PCR at anatomical sites responsible for the production of NGF supplied to DRG neurons. The lumbar spinal cord (b) and hindpaw skin (c) NGF mRNA were found decreased four weeks after diabetes induction, and the three-week treatment with EA did not affecte STZ-induced NGF mRNA decrease. Relative NGF gene expression values were calculated using the 2 −ΔΔCt method and the GAPDH used for normalization. Data are expressed as mean 2 −ΔΔCt ± SD; n = 10 for each experimental group. * P < 0.05 versus Control group.

Article Snippet: NGF in DRG extracts ( n = 10 for each experimental group) was measured by commercial ELISA (DY556, R&D Systems, MN, USA), following manufacturer's instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Increased Nerve Growth Factor Signaling in Sensory Neurons of Early Diabetic Rats Is Corrected by Electroacupuncture

doi: 10.1155/2013/652735

Figure Lengend Snippet: Effects of electroacupuncture on NGF receptors TrkA and p75 NTR in the DRGs of diabetic rats. Relative TrkA (a) and p75 NTR (b) gene expression was analyzed in DRGs by real-time PCR and data obtained using the 2 −ΔΔCt method. STZ induced a nonsignificant increase of TrkA mRNA in the DRGs (a) that reached a significant value after EA. STZ induced a significant increase of p75 NTR mRNA in the DRGs (b), while EA did not affect such increase. Data presented in (a) and (b) are expressed as mean 2 −ΔΔCt ± SD; n = 10 for each experimental group; * P < 0.05 versus control group. Representative TrkA and p Tyr496 -TrkA (p-TrkA) Western blots of three samples for each experimental group are presented (c). Densitometry of three separate gels run/blots (c) revealed that four weeks after diabetes induction, both the high-affinity NGF receptor TrkA and its activated form, the p Tyr496 -TrkA, in DRGs were increased. EA in diabetic animals induced a significant deactivation of the TrkA receptor, as indicated by the significant decrease of the phospho-TrkA in the STZ + EA group. Representative p75 NTR Western blot of three samples for each experimental group is also presented (d). Densitometry of three separate gels run/blots (d) revealed that STZ induced a significant increase of the p75 NTR in DRGs. EA reduced p75 NTR protein content in diabetic DRGs well below control level. Data presented in (c) and (d) are obtained after normalization with GAPDH bands. Western blots data ( n = 9) are all expressed as % of controls mean ± SD; * P < 0.05 versus Control group. # P < 0.05 versus STZ group.

Article Snippet: NGF in DRG extracts ( n = 10 for each experimental group) was measured by commercial ELISA (DY556, R&D Systems, MN, USA), following manufacturer's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Increased Nerve Growth Factor Signaling in Sensory Neurons of Early Diabetic Rats Is Corrected by Electroacupuncture

doi: 10.1155/2013/652735

Figure Lengend Snippet: Variations of downstream NGF signaling after STZ and EA in DRGs. Representative Western blot of three samples for each experimental group is presented in each panel, together with data from densitometry analysis of three separate gel/blot runs ( n = 9). The analysis of the total ERK1/2 and phospho Thr218-Tyr220 -ERK1/2 (p-ERK1/2) (a) and the total Akt and phospho Thr308 -Akt (p-Akt) (b) revealed that any significant variation in expression and the activation of these two TrkA-related downstream signaling kinases was induced by experimental treatments in the DRGs. The JNK that is known to be part of the p75 NTR downstream signaling machinery (c) was decreased four weeks after STZ, while phospho Thr183-Tyr185 -JNK (p-JNK) was increased. EA normalized both variations. The presence of the p75 NTR downstream signaling molecule and phosphorylated NF- κ B-p65 complex—representing an index of the nuclear translocation activity of the transcription factor NF- κ B—was unaffected by STZ (d), while EA greatly enhanced phospho-NF- κ B-p65 presence in DRGs of diabetic rats, suggesting an augmented activity of the factor. Coherently, STZ lowered the phosphorylation of the I κ B- α below controls level, suggesting an increase of its repressive activity upon NF- κ B (d); EA in diabetic rats counteracted such an effect, significantly improving I κ B- α phosphorylation versus STZ group, further indicating a decreased repression of NF- κ B activity induced by EA. Data presented in ((a)–(d)) are percent variations from the mean of control group, obtained after normalization with GAPDH band integrated optical density. Data are expressed as % of the mean of controls ( n = 9) ± SD * P < 0.05 versus control group. # P < 0.05 versus STZ group.

Article Snippet: NGF in DRG extracts ( n = 10 for each experimental group) was measured by commercial ELISA (DY556, R&D Systems, MN, USA), following manufacturer's instructions.

Techniques: Western Blot, Expressing, Activation Assay, Translocation Assay, Activity Assay, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Increased Nerve Growth Factor Signaling in Sensory Neurons of Early Diabetic Rats Is Corrected by Electroacupuncture

doi: 10.1155/2013/652735

Figure Lengend Snippet: Schematic representation of NGF system activation following STZ and EA in the sensory circuitry linking a peripheral organ (the skin), the sensory neurons located in DRG and their central innervation structure (the spinal cord). The represented mechanism is drawn on the basis of experimental results shown in the present and our previous papers. The DRG neurons receive NGF as a trophic support from both innervation fields. NGF increases throughout the entire circuitry in the first 4 weeks after STZ injection. This NGF increased availability corresponds to the NGF overactivity mediated by TrkA receptor in the spinal cord and by p75 NTR in the skin. As a result, both NGF and NGF receptors are overexpressed in the DRG neurons, with a prevalent activation of NGF receptor signaling known to be apoptosis-related and by enhanced presence and possibly activity of the NGF-regulated ion channel TRPV1, known to trigger hyperalgesia as well as neuronal sufferance (see ). Electroacupuncture regulates the activity-dependent NGF synthesis and functions, restores basal nociception and decreases NGF content in the spinal cord and DRG, normalizes TrkA activation in the spinal cord as well as p75 NTR presence in the skin and counteracts the STZ-induced events, triggered by JNK/p38 and TRPV1 (possibly a commitment of sensory neurons to apoptosis and neuronal sufferance due to calcium overload, resp.).

Article Snippet: NGF in DRG extracts ( n = 10 for each experimental group) was measured by commercial ELISA (DY556, R&D Systems, MN, USA), following manufacturer's instructions.

Techniques: Activation Assay, Injection, Activity 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: Recovery of NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Enzyme-linked Immunosorbent 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: NGF and proNGF spiked into brain cortex extract of proNGF#72 Transgenic Mouse assayed by Emax Promega with mAb Promega and mAb αD11 .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Concentration Assay, Standard Deviation

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. . 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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Mass Spectrometry, Recombinant, Comparison

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 < 0.001.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

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., ) 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., ), 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., ), 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., ), mAb αD11 (Cattaneo et al., ), 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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

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: 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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques:

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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Binding 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: Calculations of the theoretical value of the measured RU for the single NGF or proNGF components and comparison to the experimental one .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Comparison

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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Comparison, 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: proNGF CUSABIO kit: Spiking of recombinant mouse proNGF into TgProNGF#72and WT mice brain samples .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Recombinant, Standard Deviation

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: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Injection