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: 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 < 0.01 for db/db vs. controls. e Immunohistochemistry of NGF in control and db/db mouse liver. NGF immunoreactivity was present in hepatocytes and in cells adjacent to the liver sinusoids. Scale bar, 100 μm. Liver tissues from control and genetically obese mice were processed for immunoblotting and immunohistochemistry as described in Materials and methods. For immunoblotting, β-actin was used as a loading control

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, Immunohistochemistry

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: 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 < 0.01 and * p < 0.05 for treated vs. control cells. c , d SREBP2. Cells were treated with 10 ng/ml pro-NGF in the absence or presence of 0.5 μM PF429242 (PF), which is a selective inhibitor of the Site 1 protease (S1P). c Immunoblots and d quantification. PF decreased the amount of processed SREBP2 in these cells; however, addition of pro-NGF increased the relative level of cleaved SREBP2. Values are mean ± SD, n = 3. * p < 0.05 for treated vs. the corresponding control

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: 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 < 0.05 for pro-NGF vs. control and ** p < 0.01 for SB + pro-NGF vs. pro-NGF. g Mutant Flag-Thr180A-CASP2 (T180A) and Flag-Thr180E-CASP2 (T180E) caspase-2 constructs were generated by site-directed mutagenesis and transfected for 24 h into Huh7 cells in conjunction with the LDLR promoter construct to measure gene activity. Control cells expressed EGFP plasmid. Half of the cells was then stimulated with 10 ng/ml pro-NGF for 16 h. Luciferase activity was measured as described and normalized to Renilla readout. Expression of mutant T180A construct inhibited the effect of pro-NGF on LDLR, while the T180E construct increased LDLRs in controls. pro-NGF values are mean ± SD, n = 4. *** p < 0.001 for pro-NGF vs. unstimulated controls, and for T180A + pro-NGF vs. pro-NGF. * p < 0.05 for T180E unstimulated vs. controls. h Cells transfected with control GFP or mutant T180A-CASP2 construct were further stimulated with 10 ng/ml pro-NGF for 16 h. The amount of cleaved caspase-3 (17 kDa band, CASP3) and of SREBP2 (60 kDa band) was induced by pro-NGF and reduced in the presence of T180A. Expression of T180A construct is shown using anti-Flag antibodies. i Cell lysates from wild type and mutant caspase-2-expressing cells were subjected to co-immunoprecipitation (IP) using anti-Flag antibodies as described in Materials and methods. Immunoblotting was done using anti-caspase-3 antibodies. Lane 1, wild-type caspase-2; Lane 2, CARD domain lacking caspase-2 mutant; Lane 3, Ser157A caspase-2 mutant; Lane 4, Thr180A caspase-2 mutant

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, Western Blot, Mutagenesis, Construct, Generated, Transfection, Activity Assay, Plasmid Preparation, Luciferase, Expressing, Immunoprecipitation

Journal: Scientific Reports

Article Title: Development and validation of an in vitro model system to study peripheral sensory neuron development and injury

doi: 10.1038/s41598-018-34280-3

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: NTRK2 , TRKB , 1:100 , Rabbit IgG , Alomone Labs , ANT019.

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: 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