Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects P2X3 receptor-mediated currents. A, Representative examples of currents induced by α,β-meATP (black bar) on TG neurons cultured in control condition (left), after treatment with anti-NGF antibody (24 h, middle) or incubated with NGF (50 ng/ml, 24 h). B, Dose–response curves for α,β-meATP in control condition (filled circles; n = 16–24), after anti-NGF antibody treatment (filled triangles; n = 8–24; *p = 0.03), and after NGF treatment (open circles; n = 12; *p = 0.033). Current amplitudes (ΔIN) were normalized with respect to the value obtained with 10 μm α,β-meATP; p values were calculated with absolute values. Note that, despite the effect of NGF or anti-NGF antibody application on α,β-meATP-induced current amplitude, there is no difference in agonist EC50 values. C, Histogram of real-time RT-PCR experiments of TG culture mRNAs shows that NGF increases P2X3 neosynthesis (n = 3 experiments; *p = 0.03). D, Example of Western immunoblots of P2X3 receptor from total extracts of TG neurons in control (lane 1), in anti-NGF antibody (24 h; lane 2), or NGF (50 ng/ml, 24 h; lane 3). Bottom lanes show control loading with β-tubulin III. Histograms show P2X3 optical density values expressed in AUs in the different culture conditions normalized with respect to the β-tubulin III signal (mean values in AUs; n = 5 experiments; p > 0.05).

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects desensitization of currents induced by α,β-meATP. A, Representative examples of currents induced by applications of α,β-meATP (black bar) in control condition (left), after anti-NGF antibody treatment (middle), and after NGF treatment (right). Traces of 10 μm α,β-meATP-evoked currents recorded after a second agonist application (30 s interval; see arrows) are superimposed. Note the differential recovery from desensitization in the three conditions. B, Histograms show no change in the time constant values of the α,β-meATP-evoked current decay, used as an index of desensitization onset (τfast; n = 151, n = 56, and n = 46 for control, anti-NGF, and NGF, respectively). C, Histograms show that anti-NGF treatment reduces recovery (*p < 0.0001) from desensitization of P2X3-mediated currents, whereas NGF increases (*p < 0.001) this value (n = 99, n = 49, and n = 38 for control, anti-NGF, and NGF, respectively). D, Representative traces of Ca2+ transients show that, with paired α,β-meATP application (black dots, 30 s spaced), the second response is more depressed after anti-NGF treatment than in control. Neurons are identified by their responsiveness to KCl (50 mm, 1 s), which remains unchanged after anti-NGF treatment. E, Histograms showing the depressant action of anti-NGF treatment on the amplitude of Ca2+ transients induced by 10 μm α,β-meATP or 50 mm KCl. Control level is indicated by a dashed line. *p ≤ 0.05; n = 4–5 experiments (30–40 cells in each experiment).

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Phosphorylation state of P2X3 receptors is controlled by NGF. A, Example of P2X3 receptor immunoprecipitation (IP) detected in Western blot (WB) with anti-phospho-threonine antibody. P2X3 receptors from untreated TG cultures show constitutive threonine phosphorylation (lane 1). Anti-NGF-treated samples show a reduced level of P2X3 threonine phosphorylation (lane 2), which is restored after acute application of NGF (50 ng/ml, <15 min) (lane 3). Total P2X3 inputs derived from the same lysates and immunostained with anti-P2X3 antibody are also shown (bottom lanes). B, Histograms show mean values (optical density AUs) of phosphorylated P2X3 subunits obtained from anti-NGF (*p= 0.008; n = 8 experiments) or anti-NGF plus NGF (*p= 0.009; n = 8) experiments. C, Acute enhancement by NGF (50 ng/ml) of α,β-meATP-induced currents (top records) is prevented by the PKC inhibitor chelerythrine (1.5 μm in the patch pipette; bottom records). D, Histograms of changes in P2X3 subunit threonine phosphorylation after acute application of NGF (*p = 0.036; n = 9)

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Immunoprecipitation, Western Blot, Derivative Assay, Transferring

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Anti-NGF treatment increases expression of heteromeric P2X2/3 receptors. A, Histograms showing residual current amplitude in control (n = 187) and after anti-NGF (n = 59) or NGF (n = 70) treatment. *p < 0.05. B, Cumulative probability plot of the ratio Iresidual/Ipeak for control condition (filled circles; n = 224), anti-NGF antibody treatment (filled triangles; n = 79), and NGF treatment (open circles; n = 85). Note that antibody-treated, but not NGF-treated, neurons show larger Iresidual, indicative of higher contribution by heteromeric P2X2/3 receptors. The activity of heteromeric P2X2/3 receptors is estimated as the ratio between the amplitude of the steady-state Iresidual at the end of α,β-meATP application and the one of the peak current (Ipeak). C, Example of biotinylation experiments showing that NGF deprivation treatment increases the surface expression of the P2X2 subunit (top), although it does not affect the amount of surface P2X3 (bottom). Histograms indicate changes in P2X2 (black) or P2X3 (white) surface receptors measured with optical density values expressed in AUs (n = 6 or 3 experiments, respectively; *p = 0.005) and normalized with respect to the protein amount in control condition (dashed line). D, Confocal microscopy photographs of TG neurons in the control and after anti-NGF treatment show different distribution of P2X2 immunostaining. In the absence of NGF, TG neurons express P2X2 receptors on the cell surface or close by (2.5 ± 1.5 μm beneath the surface; n = 15). Scale bar, 10 μm. Histograms show the relative percentage of neurons showing diffused or ring-like P2X2 immunoreactivity in control or after anti-NGF treatment (n = 469 or 211, respectively; *p < 0.001). E, Immunopurification of heteromeric P2X2/3 receptors was obtained with chemical cross-linking treatment (DTSSP) of membrane proteins. TG neuron extracts are immunoprecipitated (IP) with anti-P2X3 antibody and immunoblotted using anti-P2X2 antibody (lanes 1, 2). P2X2 subunit (64 kDa) is immunopurified from anti-NGF antibody-treated extracts (lane 2, arrowhead) and not from control (lane 1; n = 5 experiments) after DTSSP cross-linking. Anti-NGF-treated extracts, without DTSSP cross-linking, do not show any P2X2 signal (lane 3). P2X2 immunoprecipitation detects a single band (performed as control; lane 4, arrowhead). WB, Western blot. F, Anti-NGF treatment increases the fraction of TG neurons double immunopositive for P2X2 and P2X3 subunits (*p = 0.023), whereas chronic NGF application does not change this value with respect to control (n = 4).

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Expressing, Activity Assay, Confocal Microscopy, Immunostaining, Immu-Puri, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Anti-NGF treatment differentially affects the expression of P2X2 subunit splice variants. A, Schematic representation of different P2X2 splicing forms. P2X2b or P2X2e splice variants lack 69 (from Val383 to Gln451) or 90 (Val383 to Gln472) amino acids, respectively (dashed line) in the C-terminal intracellular domain. Transmembrane regions (TM1 and TM2) are represented in black. B, Photographs of ethidium bromide-stained agarose gel electrophoresis of end-point RT-PCR experiments show the presence of P2X2 splicing forms in TG cultures grown in control conditions (lane 1) or after anti-NGF treatment (lane 2). Primers selective for the P2X2e amplify only this splicing variant in cultures treated with anti-NGF (lane 3) as well as in TG tissue (ganglion, lane 4). Amplicons show the expected length: 686, 479, and 416 bp (for P2X2a, P2X2b, and P2X2e, respectively). C, Quantitative real-time RT-PCR experiments performed with P2X2 splicing-specific primers. Anti-NGF antibody treatment upregulates P2X2b and P2X2e mRNA levels (n = 3; *p = 0.04 and *p = 0.02, respectively), although it proportionally reduces P2X2a (n = 3; *p = 0.04). Chronic NGF treatment has no effect (left histograms). Samples are normalized with respect to mRNA levels in control condition (dashed line). D, Deglycosylation experiment of P2X2 subunit in total extract of TG cultures in control or after anti-NGF treatment (lanes 1, 2; n = 4). The same protocol is also applied to P2X2/3 receptors immunopurified from anti-NGF-treated cells (lane 5; n = 2). Lane 3 shows untreated culture extract without PNGaseF application; lane 4 shows single band (P2X2/3 receptor) without PNGaseF treatment from culture after anti-NGF. Note that PNGaseF deglycosylation (PNG) is required to recognize discrete double bands (with apparent molecular weight of 50 and 45 kDa, respectively; lanes 2 and 5, arrows) relative to P2X2 differentially spliced polypeptides. IP, Immunoprecipitation; WB, Western blot.

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Expressing, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Quantitative RT-PCR, Molecular Weight, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: The potentiating action of NGF on P2X3 receptor-mediated responses does not depend on CGRP. A, Examples of currents induced by α,β-meATP in TG neurons grown in control condition, in the presence of NGF (50 ng/ml, 24 h), in the presence of CGRP receptor antagonist BIBN4096BS (BIBN; 50 ng/ml, 24 h), or with NGF plus BIBN4096BS (24 h). B, Somatic size distribution of TG neurons immunostained with TrkA receptor antibody (filled columns) and labeled with CGRP-RITC (open columns) to investigate colocalization of NGF and CGRP receptors (filled, cross-hatched columns). Data are from ∼1000 cells (n = 3 independent experiments). IR, Immunoreactivity. C, Example of patch-clamp traces showing that CGRP application (1 μm, 1 h) to anti-NGF antibody-treated neurons increases α,β-meATP responses and accelerates recovery from desensitization (30 s interval between twin pulses). D, CGRP (1 μm, 1 h) potentiates α,β-meATP-evoked currents in control condition (n = 19; *p < 0.0001) and after anti-NGF treatment (n = 18; *p < 0.0001).

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Labeling, Patch Clamp

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Trigeminal nociception evoked by α,β-meATP is prevented by anti-NGF treatment. A, Histograms showing the time spent by mice in face-rubbing activity after subcutaneous injection of α,β-meATP (10 μl bolus of 10 mm solution) into the upper lip after pretreatment with anti-NGF or saline (sal). Each bar is the mean of six to eight animals observed for the first 15 min epoch. No significant changes in rubbing activity are observed during this period. All mice were pretreated 24 h earlier with saline (10 μl/g, i.p.) or anti-NGF antibody (300 ng/g, i.p.). B, In the 15–30 min epoch, a significant increase (*p = 0.005) in rubbing activity is observed after injection of α,β-meATP (n = 8). This algogenic effect of α,β-meATP is blocked (*p= 0.025 vs sham pretreatment) after anti-NGF pretreatment (n = 7).

Article Snippet: To exclude cross-reactivity of anti-NGF antibodies with other neurotrophins, the dot blot of NGF, BDNF, glial cell line-derived neurotrophic factor, and neurotrophins 3 and 4 (50 ng; Alomone Labs or Sigma) was immunostained with anti-NGF antibody (1:2000; Sigma), showing staining for NGF only and nonspecific signals.

Techniques: Activity Assay, Injection

Journal: Journal of diabetes, metabolic disorders & control

Article Title: Deletion of the Neurotrophin Receptor p75 NTR Prevents Diabetes-Induced Retinal Acellular Capillaries in Streptozotocin-Induced Mouse Diabetic Model

doi: 10.15406/jdmdc.2017.04.00129

Figure Lengend Snippet: A summary of sources of antibodies used to detect protein expression using Western Blot.

Article Snippet: Band intensity was quantified using densitometry software (alphEaseFC) and normalized to β-actin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Catalog Number Company Anti-VEGF Cat.# 676472 Millipore, Billerica, MA Anti-EFNB2 Cat #: PA5-19361 Anti-proNGF Cat.# N-250 Alomone Labs, Jerusalem, Israel Anti-NGF Cat.# AN-240 Anti-IL-1β Cat.# ab9722 Abcam, Cambridge, MA Anti-GAPDH Cat.# G8795 Sigma-Aldrich, St. Louis, MO Anti-b-actin Cat.# A2228 Anti-JNK Cat.# sc-7345 Santa Cruz Biotechnology, Dallas, Texas Anti-p-JNK Cat.# sc-293136 Cleaved-PARP Cat.# 5625 Cell Signaling Technology, Danvers, MA Anti-p75 NTR ## Dr. Bruce Carter (Dept. of Biochemistry, Vanderbilt University Open in a separate window GAPDH: Glyceraldehyde 3-phosphate Dehydrogenase; IL-1 β: Interleukin-1 beta; JNKC: June Kinase; NGF: Nerve Growth Factor; proNGF: Proform of Nerve Growth Factor; p75 NTR : Neurotrophin Receptor p75; TrkA: Tyrosine Kinase Receptor-A; VEGF: Vascular Endothelial Growth Factor A summary of sources of antibodies used to detect protein expression using Western Blot.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Expression levels of VEGF-C and VEGFR-3 in renal cell carcinoma and their association with lymph node metastasis

doi: 10.3892/etm.2021.9986

Figure Lengend Snippet: Immunohistochemical staining of VEGFR-3 expression. VEGFR-3 immunohistochemical staining in (A) normal renal tissues and (B) RCC tissues. (C) Lymphatic endothelial cells in RCC tissues were VEGFR-3 + (indicated by arrows). Scale bars, 100 µm. (D) Correlation between VEGF-C and VEGFR-3 expression in RCC tissues. Statistical significance was determined by Spearman's correlation test. RCC, renal cell carcinoma; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, VEGF receptor-3.

Article Snippet: For antigen retrieval, the tissue sections were incubated with 0.01 M sodium citrate (pH 6) in a microwave oven at 95˚C for 10 min, followed by blocking with 5% normal goat serum (cat. no. ZLI-9021; OriGene Technologies, Inc.) for 10 min at room temperature, the tissue sections were incubated with rabbit anti-human VEGF-C monoclonal antibody (cat. no. BA0548; 1:200; Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human VEGFR-3 monoclonal antibody (cat. no. A01276-3; 1:200; Wuhan Boster Biological Technology Co., Ltd.) and mouse anti-human D2-40 monoclonal antibody (cat. no. ZM-0465; undiluted; OriGene Technologies, Inc.) for 12 h at 4˚C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: iScience

Article Title: Kidney fibrosis molecular mechanisms Spp1 influences fibroblast activity through transforming growth factor beta smad signaling

doi: 10.1016/j.isci.2024.109839

Figure Lengend Snippet:

Article Snippet: TGF-β1 , Cell Signaling, USA , 5231LF.

Techniques: Recombinant, Staining, CCK-8 Assay, Enzyme-linked Immunosorbent Assay