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    Alomone Labs ngf
    Foretinib and lestaurtinib inhibit the low levels of TrkA tyrosine phosphorylation in <t>NGF-deprived</t> neurons and suppress axon degeneration. (A) Quantitative normalized representation of the phosphoproteomics analysis performed on rat sensory neurons in the indicated conditions for 8 h, showing the relative amount of TrkA peptides containing phosphorylated Y499 (Shc binding site) or Y683 and Y684 (activation loop tyrosines). (B) WB analysis of mouse sympathetic neurons withdrawn from <t>NGF</t> with or without 500 nM foretinib (Foret) or 1 µM Lestaurtinib (Lest) for 10 h, probed for TrkA phosphorylated at Y683/Y684, and reprobed for total TrkA. (C) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of 1 µM lestaurtinib for 24 h and immunostained for βIII-tubulin (red) and counterstained with Hoechst 33258 (blue; bottom) and bright-field (top). The arrow indicates neuritic beading and the arrowhead an apoptotic cell. (D and E) Images as in C quantified for the number of swellings or beads (D) and the ratio of intact neurites (E). **, P
    Ngf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria"

    Article Title: A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201705085

    Foretinib and lestaurtinib inhibit the low levels of TrkA tyrosine phosphorylation in NGF-deprived neurons and suppress axon degeneration. (A) Quantitative normalized representation of the phosphoproteomics analysis performed on rat sensory neurons in the indicated conditions for 8 h, showing the relative amount of TrkA peptides containing phosphorylated Y499 (Shc binding site) or Y683 and Y684 (activation loop tyrosines). (B) WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib (Foret) or 1 µM Lestaurtinib (Lest) for 10 h, probed for TrkA phosphorylated at Y683/Y684, and reprobed for total TrkA. (C) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of 1 µM lestaurtinib for 24 h and immunostained for βIII-tubulin (red) and counterstained with Hoechst 33258 (blue; bottom) and bright-field (top). The arrow indicates neuritic beading and the arrowhead an apoptotic cell. (D and E) Images as in C quantified for the number of swellings or beads (D) and the ratio of intact neurites (E). **, P
    Figure Legend Snippet: Foretinib and lestaurtinib inhibit the low levels of TrkA tyrosine phosphorylation in NGF-deprived neurons and suppress axon degeneration. (A) Quantitative normalized representation of the phosphoproteomics analysis performed on rat sensory neurons in the indicated conditions for 8 h, showing the relative amount of TrkA peptides containing phosphorylated Y499 (Shc binding site) or Y683 and Y684 (activation loop tyrosines). (B) WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib (Foret) or 1 µM Lestaurtinib (Lest) for 10 h, probed for TrkA phosphorylated at Y683/Y684, and reprobed for total TrkA. (C) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of 1 µM lestaurtinib for 24 h and immunostained for βIII-tubulin (red) and counterstained with Hoechst 33258 (blue; bottom) and bright-field (top). The arrow indicates neuritic beading and the arrowhead an apoptotic cell. (D and E) Images as in C quantified for the number of swellings or beads (D) and the ratio of intact neurites (E). **, P

    Techniques Used: Binding Assay, Activation Assay, Western Blot

    Foretinib inhibits local sympathetic axon degeneration caused by trophic factor deprivation. (A) Rat sympathetic neurons were switched into the indicated conditions for 48 h and immunostained for βIII-tubulin (white). Schematics on the left show the configurations used. In the top panels (i), NGF was maintained in the center compartment and removed from both side compartments, and 500 nM foretinib (Foret) added to one of the sides. In the center and bottom panels (ii and iii), NGF was withdrawn from all compartments, and foretinib added to one of the sides (ii) or one of the sides and the center compartment (iii). Fluorescence images show βIII-tubulin–positive axons in the two sides of the same compartments, and the bright-field images the cell bodies and proximal axons in the center compartments of the same cultures. Arrowhead denotes a shrunken, dying neuron. (B) Number of swellings or beads per 100 µm of neurite in the side compartments withdrawn from NGF with and without 500 nM foretinib. **, P
    Figure Legend Snippet: Foretinib inhibits local sympathetic axon degeneration caused by trophic factor deprivation. (A) Rat sympathetic neurons were switched into the indicated conditions for 48 h and immunostained for βIII-tubulin (white). Schematics on the left show the configurations used. In the top panels (i), NGF was maintained in the center compartment and removed from both side compartments, and 500 nM foretinib (Foret) added to one of the sides. In the center and bottom panels (ii and iii), NGF was withdrawn from all compartments, and foretinib added to one of the sides (ii) or one of the sides and the center compartment (iii). Fluorescence images show βIII-tubulin–positive axons in the two sides of the same compartments, and the bright-field images the cell bodies and proximal axons in the center compartments of the same cultures. Arrowhead denotes a shrunken, dying neuron. (B) Number of swellings or beads per 100 µm of neurite in the side compartments withdrawn from NGF with and without 500 nM foretinib. **, P

    Techniques Used: Fluorescence

    Foretinib suppresses the expression or activity of genes and proteins associated with sympathetic neuron death and degeneration and protects mitochondria. (A and B) Changes in gene expression in mouse sympathetic neurons withdrawn from NGF for 9 h ( bimEL , puma/bbc3 , trib3 , ddit3 , and hrk mRNAs) or 12 h ( pmaip1/noxa , gadd45γ , tap63 mRNAs) with or without foretinib (Foret) by quantitative RT-PCR; n ≥ 3 independent experiments. (C–G). WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib for 10–12 (C and F), 18 (D and E), or 24 (G) h. Blots were probed with antibodies to the indicated proteins, including phosphorylated (p) JNK, activated (a) Bax, cleaved caspase-3 (cc3), or αII-spectrin (cleaved [cl] and full-length [f.l.]), and reprobed for βIII-tubulin (tuj1), ERK1/2, or GAPDH to control for loading. (H and I) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of foretinib for 12 h, immunostained for cytochrome c (red, cyt-c) or βIII-tubulin (green), and quantified for neurons with diffuse cytochrome c (I), as indicated by arrows in H. (I) ***, P
    Figure Legend Snippet: Foretinib suppresses the expression or activity of genes and proteins associated with sympathetic neuron death and degeneration and protects mitochondria. (A and B) Changes in gene expression in mouse sympathetic neurons withdrawn from NGF for 9 h ( bimEL , puma/bbc3 , trib3 , ddit3 , and hrk mRNAs) or 12 h ( pmaip1/noxa , gadd45γ , tap63 mRNAs) with or without foretinib (Foret) by quantitative RT-PCR; n ≥ 3 independent experiments. (C–G). WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib for 10–12 (C and F), 18 (D and E), or 24 (G) h. Blots were probed with antibodies to the indicated proteins, including phosphorylated (p) JNK, activated (a) Bax, cleaved caspase-3 (cc3), or αII-spectrin (cleaved [cl] and full-length [f.l.]), and reprobed for βIII-tubulin (tuj1), ERK1/2, or GAPDH to control for loading. (H and I) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of foretinib for 12 h, immunostained for cytochrome c (red, cyt-c) or βIII-tubulin (green), and quantified for neurons with diffuse cytochrome c (I), as indicated by arrows in H. (I) ***, P

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot

    Foretinib prevents NGF-induced death and degeneration of sympathetic and sensory neurons. (A–D) Foretinib protects sympathetic neurons from degeneration more effectively than a JNK inhibitor. Murine sympathetic neurons were cultured in NGF for 2 d, withdrawn from NGF (−NGF), and cultured for 16 h with or without 2 µM JNK inhibitor TAT-TI-JIP 153–156 (JNKiVII), 500 nM foretinib (Foret), or NGF. Shown is immunostaining for βIII-tubulin (red) and costaining with Hoechst 33258 to highlight nuclear morphology (bottom) or bright-field (top) panels. Images were quantified as described in Materials and methods for the number of intact neurites (B), axonal beads (C), or condensed apoptotic nuclei (D). *, P
    Figure Legend Snippet: Foretinib prevents NGF-induced death and degeneration of sympathetic and sensory neurons. (A–D) Foretinib protects sympathetic neurons from degeneration more effectively than a JNK inhibitor. Murine sympathetic neurons were cultured in NGF for 2 d, withdrawn from NGF (−NGF), and cultured for 16 h with or without 2 µM JNK inhibitor TAT-TI-JIP 153–156 (JNKiVII), 500 nM foretinib (Foret), or NGF. Shown is immunostaining for βIII-tubulin (red) and costaining with Hoechst 33258 to highlight nuclear morphology (bottom) or bright-field (top) panels. Images were quantified as described in Materials and methods for the number of intact neurites (B), axonal beads (C), or condensed apoptotic nuclei (D). *, P

    Techniques Used: Cell Culture, Immunostaining

    TrkA inhibition partially rescues sympathetic neuron degeneration. (A) WB analysis of TrkA F592A sympathetic neurons withdrawn from NGF in the presence of 1 µM 1NMPP1 for 10 h, probed for Trk phosphorylated at Y683/Y684, and reprobed for total TrkA. (B) The specific TrkA F592A inhibitor 1NMPP1 partially rescues NGF withdrawal-induced degeneration. TrkA F592A sympathetic neurons cultured withdrawn from NGF in the presence or absence of 1 µM 1NMPP1 for 14 h and immunostained for βIII-tubulin and counterstained with Hoechst 33258. (C and D) Images as in B were quantified for the number of swellings or beads or for the ratio of intact neurites. (C) *, P
    Figure Legend Snippet: TrkA inhibition partially rescues sympathetic neuron degeneration. (A) WB analysis of TrkA F592A sympathetic neurons withdrawn from NGF in the presence of 1 µM 1NMPP1 for 10 h, probed for Trk phosphorylated at Y683/Y684, and reprobed for total TrkA. (B) The specific TrkA F592A inhibitor 1NMPP1 partially rescues NGF withdrawal-induced degeneration. TrkA F592A sympathetic neurons cultured withdrawn from NGF in the presence or absence of 1 µM 1NMPP1 for 14 h and immunostained for βIII-tubulin and counterstained with Hoechst 33258. (C and D) Images as in B were quantified for the number of swellings or beads or for the ratio of intact neurites. (C) *, P

    Techniques Used: Inhibition, Western Blot, Cell Culture

    2) Product Images from "Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway"

    Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-15-108

    PI3K activity regulates the constitutive levels of ApoER2 CTF but is not involved in ApoER2 shedding induced by NGF. (A) PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and 50 μM LY294002 or 5 μM ZSTK474 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2 and p75 NTR were recognized using antibodies directed against their intracellular regions. The activation of PI3K, induced by NGF, was determined by detection of phospho-AKT. α-tubulin is shown as a loading control. The blot levels of ApoER2 CTF (B and C) and of p75 NTR CTF (D and E) were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, * P
    Figure Legend Snippet: PI3K activity regulates the constitutive levels of ApoER2 CTF but is not involved in ApoER2 shedding induced by NGF. (A) PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and 50 μM LY294002 or 5 μM ZSTK474 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2 and p75 NTR were recognized using antibodies directed against their intracellular regions. The activation of PI3K, induced by NGF, was determined by detection of phospho-AKT. α-tubulin is shown as a loading control. The blot levels of ApoER2 CTF (B and C) and of p75 NTR CTF (D and E) were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, * P

    Techniques Used: Activity Assay, Incubation, Activation Assay

    3) Product Images from "Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation"

    Article Title: Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100926

    TrkA activation is modulated by p75-TMD. A , protein sequences of the different mutant constructs of p75-TMD. The residue mutated is shown in bold . B , western blots of lysates from HeLa cells transfected with the indicated constructs and stimulated with increasing concentrations of NGF. Membranes were probed using a TrkA-P-Tyr675 specific antibody. C , normalized activation of TrkA using increasing concentrations of NGF in the absence or the presence of p75 mutant constructs indicated. The bars represent the standard error of at least three independent experiments. p Values are reported in the article. NGF, nerve growth factor; TMD, transmembrane domain; TrkA, tyrosine potein kinase receptor A.
    Figure Legend Snippet: TrkA activation is modulated by p75-TMD. A , protein sequences of the different mutant constructs of p75-TMD. The residue mutated is shown in bold . B , western blots of lysates from HeLa cells transfected with the indicated constructs and stimulated with increasing concentrations of NGF. Membranes were probed using a TrkA-P-Tyr675 specific antibody. C , normalized activation of TrkA using increasing concentrations of NGF in the absence or the presence of p75 mutant constructs indicated. The bars represent the standard error of at least three independent experiments. p Values are reported in the article. NGF, nerve growth factor; TMD, transmembrane domain; TrkA, tyrosine potein kinase receptor A.

    Techniques Used: Activation Assay, Mutagenesis, Construct, Western Blot, Transfection

    TrkA–p75 FSI–FRET experiments. A , illustrations of the TrkA-mTurq and p75-eYFP proteins used in FRET experiments along with the LAT-mTurq and ECTM FGFR3–eYFP proteins used in control experiments. B , FRET efficiencies as a function of total receptor concentration measured for TrkA-mTurq and p75-eYFP in the absence of ligand compared with a zero FRET control dataset. C , illustrations of some possible stoichiometries of the TrkA–p75 heterocomplex: (i) heterodimer, (ii) heterotrimer of two TrkA and one p75, (iii) heterotrimer of one TrkA and two p75, and (iv) heterotetramer or two TrkA and two p75. D , FRET data for TrkA-mTurq and p75-eYFP in the presence of 100 ng/μl NGF compared with the data in the absence of NGF. E , the FRET data for TrkA and p75 in the presence of NGF compared with the zero FRET control dataset. F , expression of TrkA-mTurq and p75-eYFP measured on the cell surface for the experiments performed in the absence and presence of NGF. G , the FRET data for TrkA–p75 in the absence and presence of NGF and for the control dataset were binned and compared. H , illustrations of the possible consequences of NGF binding to the TrkA–p75 heterocomplex, which could be either dissociation of the heterocomplex to stabilize the respective homodimers or an NGF-induced conformational change. The bars represent the standard error of the mean. eYFP, enhanced YFP; FGFR3, fibroblast growth factor receptor 3; FSI, fully quantified spectral imaging; LAT, linker for the activation of T-cells; mTurq, mTurquoise; NGF, nerve growth factor; TrkA, tyrosine protein kinase receptor A.
    Figure Legend Snippet: TrkA–p75 FSI–FRET experiments. A , illustrations of the TrkA-mTurq and p75-eYFP proteins used in FRET experiments along with the LAT-mTurq and ECTM FGFR3–eYFP proteins used in control experiments. B , FRET efficiencies as a function of total receptor concentration measured for TrkA-mTurq and p75-eYFP in the absence of ligand compared with a zero FRET control dataset. C , illustrations of some possible stoichiometries of the TrkA–p75 heterocomplex: (i) heterodimer, (ii) heterotrimer of two TrkA and one p75, (iii) heterotrimer of one TrkA and two p75, and (iv) heterotetramer or two TrkA and two p75. D , FRET data for TrkA-mTurq and p75-eYFP in the presence of 100 ng/μl NGF compared with the data in the absence of NGF. E , the FRET data for TrkA and p75 in the presence of NGF compared with the zero FRET control dataset. F , expression of TrkA-mTurq and p75-eYFP measured on the cell surface for the experiments performed in the absence and presence of NGF. G , the FRET data for TrkA–p75 in the absence and presence of NGF and for the control dataset were binned and compared. H , illustrations of the possible consequences of NGF binding to the TrkA–p75 heterocomplex, which could be either dissociation of the heterocomplex to stabilize the respective homodimers or an NGF-induced conformational change. The bars represent the standard error of the mean. eYFP, enhanced YFP; FGFR3, fibroblast growth factor receptor 3; FSI, fully quantified spectral imaging; LAT, linker for the activation of T-cells; mTurq, mTurquoise; NGF, nerve growth factor; TrkA, tyrosine protein kinase receptor A.

    Techniques Used: Concentration Assay, Expressing, Binding Assay, Imaging, Activation Assay

    Effect of the mutation of the p75 heterodimer interface. A and B , result of 12 simulations by CG-MD of p75-TMD-AGA mutant ( A ) or p75-TMD-wt ( B ) and TrkA-TMD in POPC model membranes. The position of the p75-TMD helix ( gray ) respect to the TrkA-TMD ( red ) after each simulation is shown. In green and red are shown the residues that belong to the active and inactive homodimer interface of TrkA described in the study by Franco et al. C , quantification of the neurite length (micrometer) of PC12 cells electroporated with the indicated constructs and GFP at 24 h of addition of NGF (50 ng/ml). The bars represent the standard error of at least three independent electroporation experiments. Statistical analysis was performed with one-way ANOVA, and the p values are reported above each bar. D , representative fluorescence microscopy of PC12 cells electroporated with the indicated constructs stimulated with NGF (50 ng/ml) for 24 h after electroporation. The bar represents 50 μm. CG, coarse-grained; MD, molecular dynamics; NGF, nerve growth factor; POPC, palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; TMD, transmembrane domain.
    Figure Legend Snippet: Effect of the mutation of the p75 heterodimer interface. A and B , result of 12 simulations by CG-MD of p75-TMD-AGA mutant ( A ) or p75-TMD-wt ( B ) and TrkA-TMD in POPC model membranes. The position of the p75-TMD helix ( gray ) respect to the TrkA-TMD ( red ) after each simulation is shown. In green and red are shown the residues that belong to the active and inactive homodimer interface of TrkA described in the study by Franco et al. C , quantification of the neurite length (micrometer) of PC12 cells electroporated with the indicated constructs and GFP at 24 h of addition of NGF (50 ng/ml). The bars represent the standard error of at least three independent electroporation experiments. Statistical analysis was performed with one-way ANOVA, and the p values are reported above each bar. D , representative fluorescence microscopy of PC12 cells electroporated with the indicated constructs stimulated with NGF (50 ng/ml) for 24 h after electroporation. The bar represents 50 μm. CG, coarse-grained; MD, molecular dynamics; NGF, nerve growth factor; POPC, palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; TMD, transmembrane domain.

    Techniques Used: Mutagenesis, Construct, Electroporation, Fluorescence, Microscopy

    4) Product Images from "Secreted herpes simplex virus-2 glycoprotein G alters thermal pain sensitivity by modifying NGF effects on TRPV1"

    Article Title: Secreted herpes simplex virus-2 glycoprotein G alters thermal pain sensitivity by modifying NGF effects on TRPV1

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0677-5

    SgG2 increases NGF-dependent TRPV1 serine phosphorylation. DRG neurons were grown for 3 days in NGF medium, NGF starved for 16 h, and stimulated with HEPES, NGF in HEPES, or NGF plus SgG2 for 30 min. Western blots showing phosphorylation of TrkA and p38 ( a ) and TRPV1 phosphorylation in serine residues ( b ), which was detected following TRPV1 immunoprecipitation. c Graph showing the quantified serine phosphorylation in TRPV1. The data corresponds to the average of three independent experiments for TRPV1 serine phosphorylation. Error bars represent the mean plus standard deviation * p
    Figure Legend Snippet: SgG2 increases NGF-dependent TRPV1 serine phosphorylation. DRG neurons were grown for 3 days in NGF medium, NGF starved for 16 h, and stimulated with HEPES, NGF in HEPES, or NGF plus SgG2 for 30 min. Western blots showing phosphorylation of TrkA and p38 ( a ) and TRPV1 phosphorylation in serine residues ( b ), which was detected following TRPV1 immunoprecipitation. c Graph showing the quantified serine phosphorylation in TRPV1. The data corresponds to the average of three independent experiments for TRPV1 serine phosphorylation. Error bars represent the mean plus standard deviation * p

    Techniques Used: Western Blot, Immunoprecipitation, Standard Deviation

    5) Product Images from "Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels"

    Article Title: Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1653-17.2018

    ARMS regulates BDNF release in DRG and cortical neurons. A , ARMS depletion in cultured DRG neurons. Cultured DRG neurons were infected at DIV 4 with control (shControl), ARMS shRNA-1 (shARMS-1), or ARMS shRNA-2 (shARMS-2) lentiviruses and lysates were obtained at DIV 11. Western blot analyses were performed. A representative blot is shown ( n = 4). B , BDNF secretion in response to NGF is enhanced in ARMS-depleted DRG neurons. BDNF ELISA was performed using the supernatant of DRG neurons from A that were nonstimulated (basal) and then stimulated with NGF for 30 min at DIV 11. Cell lysates were collected to assess BDNF levels ( n = 4). Paired Student's t test, mean ± SEM. shControl versus shARMS-1, t = 3.745, df = 3; shControl versus shARMS-2, t = 3.638, df = 3. C , ARMS knockdown in cultured cortical neurons. Cultured cortical neurons were infected with shControl, shARMS-1, or shARMS-2 lentiviruses at DIV 2 and with lentiviruses expressing BDNF at DIV 7. Cell lysates were obtained at DIV 10. Western blot analyses were performed. A representative blot is shown ( n = 4). D , ARMS knockdown potentiates BDNF release in response to different stimuli in cortical neurons. BDNF ELISA was performed using the supernatant of cortical neurons from C that were nonstimulated (basal) and then stimulated with KCl, NT-3, or NT-4 for 30 min at DIV 10 ( n = 9, n = 5, and n = 4 for shControl, shARMS-1, and shARMS-2, respectively). Unpaired Student's t test, mean ± SEM. K + Depol: shControl versus shARMS-1, t = 2.846, df = 12; shControl versus shARMS-2, t = 5.923, df = 11; NT-3: shControl versus shARMS-1, t = 3.917, df = 12; shControl versus shARMS-2, t = 10.03, df = 11; NT-4: shControl versus shARMS-1, t = 3.742, df = 12; shControl versus shARMS-2, t .
    Figure Legend Snippet: ARMS regulates BDNF release in DRG and cortical neurons. A , ARMS depletion in cultured DRG neurons. Cultured DRG neurons were infected at DIV 4 with control (shControl), ARMS shRNA-1 (shARMS-1), or ARMS shRNA-2 (shARMS-2) lentiviruses and lysates were obtained at DIV 11. Western blot analyses were performed. A representative blot is shown ( n = 4). B , BDNF secretion in response to NGF is enhanced in ARMS-depleted DRG neurons. BDNF ELISA was performed using the supernatant of DRG neurons from A that were nonstimulated (basal) and then stimulated with NGF for 30 min at DIV 11. Cell lysates were collected to assess BDNF levels ( n = 4). Paired Student's t test, mean ± SEM. shControl versus shARMS-1, t = 3.745, df = 3; shControl versus shARMS-2, t = 3.638, df = 3. C , ARMS knockdown in cultured cortical neurons. Cultured cortical neurons were infected with shControl, shARMS-1, or shARMS-2 lentiviruses at DIV 2 and with lentiviruses expressing BDNF at DIV 7. Cell lysates were obtained at DIV 10. Western blot analyses were performed. A representative blot is shown ( n = 4). D , ARMS knockdown potentiates BDNF release in response to different stimuli in cortical neurons. BDNF ELISA was performed using the supernatant of cortical neurons from C that were nonstimulated (basal) and then stimulated with KCl, NT-3, or NT-4 for 30 min at DIV 10 ( n = 9, n = 5, and n = 4 for shControl, shARMS-1, and shARMS-2, respectively). Unpaired Student's t test, mean ± SEM. K + Depol: shControl versus shARMS-1, t = 2.846, df = 12; shControl versus shARMS-2, t = 5.923, df = 11; NT-3: shControl versus shARMS-1, t = 3.917, df = 12; shControl versus shARMS-2, t = 10.03, df = 11; NT-4: shControl versus shARMS-1, t = 3.742, df = 12; shControl versus shARMS-2, t .

    Techniques Used: Cell Culture, Infection, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    6) Product Images from "Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels"

    Article Title: Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1653-17.2018

    ARMS regulates BDNF release in DRG and cortical neurons. A , ARMS depletion in cultured DRG neurons. Cultured DRG neurons were infected at DIV 4 with control (shControl), ARMS shRNA-1 (shARMS-1), or ARMS shRNA-2 (shARMS-2) lentiviruses and lysates were obtained at DIV 11. Western blot analyses were performed. A representative blot is shown ( n = 4). B , BDNF secretion in response to NGF is enhanced in ARMS-depleted DRG neurons. BDNF ELISA was performed using the supernatant of DRG neurons from A that were nonstimulated (basal) and then stimulated with NGF for 30 min at DIV 11. Cell lysates were collected to assess BDNF levels ( n = 4). Paired Student's t test, mean ± SEM. shControl versus shARMS-1, t = 3.745, df = 3; shControl versus shARMS-2, t = 3.638, df = 3. C , ARMS knockdown in cultured cortical neurons. Cultured cortical neurons were infected with shControl, shARMS-1, or shARMS-2 lentiviruses at DIV 2 and with lentiviruses expressing BDNF at DIV 7. Cell lysates were obtained at DIV 10. Western blot analyses were performed. A representative blot is shown ( n = 4). D , ARMS knockdown potentiates BDNF release in response to different stimuli in cortical neurons. BDNF ELISA was performed using the supernatant of cortical neurons from C that were nonstimulated (basal) and then stimulated with KCl, NT-3, or NT-4 for 30 min at DIV 10 ( n = 9, n = 5, and n = 4 for shControl, shARMS-1, and shARMS-2, respectively). Unpaired Student's t test, mean ± SEM. K + Depol: shControl versus shARMS-1, t = 2.846, df = 12; shControl versus shARMS-2, t = 5.923, df = 11; NT-3: shControl versus shARMS-1, t = 3.917, df = 12; shControl versus shARMS-2, t = 10.03, df = 11; NT-4: shControl versus shARMS-1, t = 3.742, df = 12; shControl versus shARMS-2, t .
    Figure Legend Snippet: ARMS regulates BDNF release in DRG and cortical neurons. A , ARMS depletion in cultured DRG neurons. Cultured DRG neurons were infected at DIV 4 with control (shControl), ARMS shRNA-1 (shARMS-1), or ARMS shRNA-2 (shARMS-2) lentiviruses and lysates were obtained at DIV 11. Western blot analyses were performed. A representative blot is shown ( n = 4). B , BDNF secretion in response to NGF is enhanced in ARMS-depleted DRG neurons. BDNF ELISA was performed using the supernatant of DRG neurons from A that were nonstimulated (basal) and then stimulated with NGF for 30 min at DIV 11. Cell lysates were collected to assess BDNF levels ( n = 4). Paired Student's t test, mean ± SEM. shControl versus shARMS-1, t = 3.745, df = 3; shControl versus shARMS-2, t = 3.638, df = 3. C , ARMS knockdown in cultured cortical neurons. Cultured cortical neurons were infected with shControl, shARMS-1, or shARMS-2 lentiviruses at DIV 2 and with lentiviruses expressing BDNF at DIV 7. Cell lysates were obtained at DIV 10. Western blot analyses were performed. A representative blot is shown ( n = 4). D , ARMS knockdown potentiates BDNF release in response to different stimuli in cortical neurons. BDNF ELISA was performed using the supernatant of cortical neurons from C that were nonstimulated (basal) and then stimulated with KCl, NT-3, or NT-4 for 30 min at DIV 10 ( n = 9, n = 5, and n = 4 for shControl, shARMS-1, and shARMS-2, respectively). Unpaired Student's t test, mean ± SEM. K + Depol: shControl versus shARMS-1, t = 2.846, df = 12; shControl versus shARMS-2, t = 5.923, df = 11; NT-3: shControl versus shARMS-1, t = 3.917, df = 12; shControl versus shARMS-2, t = 10.03, df = 11; NT-4: shControl versus shARMS-1, t = 3.742, df = 12; shControl versus shARMS-2, t .

    Techniques Used: Cell Culture, Infection, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    7) Product Images from "TrkA In Vivo Function Is Negatively Regulated by Ubiquitination"

    Article Title: TrkA In Vivo Function Is Negatively Regulated by Ubiquitination

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4294-13.2014

    NGF leads to increased TrkA activation and higher AKT and MEK phosphorylation in TrkAΔKFG mutant neurons. A , DRG neurons were isolated from E13.5 WT (+/+), heterozygous (+/−), or TrkAΔKFG mutant (−/−) embryos (6–8
    Figure Legend Snippet: NGF leads to increased TrkA activation and higher AKT and MEK phosphorylation in TrkAΔKFG mutant neurons. A , DRG neurons were isolated from E13.5 WT (+/+), heterozygous (+/−), or TrkAΔKFG mutant (−/−) embryos (6–8

    Techniques Used: Activation Assay, Mutagenesis, Isolation

    Lys 450 in the KFG motif is important for the ubiquitination of TrkA receptors in response to NGF. A–C , HEK293T cell lines stably expressing a single copy of WT, KFG deleted (ΔKFG), AFG (single lysine to alanine mutation), or KAA (phenylalanine
    Figure Legend Snippet: Lys 450 in the KFG motif is important for the ubiquitination of TrkA receptors in response to NGF. A–C , HEK293T cell lines stably expressing a single copy of WT, KFG deleted (ΔKFG), AFG (single lysine to alanine mutation), or KAA (phenylalanine

    Techniques Used: Stable Transfection, Expressing, Mutagenesis

    Deletion of the KFG domain from TrkA alters NGF-induced trafficking of TrkA. A , HEK293T cells stably expressing WT or KFG deleted TrkA as in were treated with 100 ng/ml NGF for the indicated time (1–6 h) followed by biotinylation. Cell
    Figure Legend Snippet: Deletion of the KFG domain from TrkA alters NGF-induced trafficking of TrkA. A , HEK293T cells stably expressing WT or KFG deleted TrkA as in were treated with 100 ng/ml NGF for the indicated time (1–6 h) followed by biotinylation. Cell

    Techniques Used: Stable Transfection, Expressing

    8) Product Images from "B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS"

    Article Title: B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131780

    Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.
    Figure Legend Snippet: Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.

    Techniques Used: Expressing, Mouse Assay

    Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.
    Figure Legend Snippet: Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.

    Techniques Used: Activation Assay, Expressing, Staining, Mouse Assay, Cell Culture, In Vitro

    9) Product Images from "Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration"

    Article Title: Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration

    Journal: Molecular Vision

    doi:

    Overexpression of proNGF reduced NGF and induced expression of TrkA and p75 NTR , but not sortilin. A , B : Western blot (WB) analysis of rat retinal lysate showed significant (1.5-fold) increase in expression of TrkA and p75 NTR in rats electroporated with pGFP-proNGF123 as compared with those electroporated with pGFP (n=4). C : WB analysis showed that overexpression of pGFP-proNGF123 in rat retinas did not affect sortilin protein expression level as compared with the control rats (n=4). D : WB analysis showed that overexpression of pGFP-proNGF123 significantly reduced NGF expression (n=5) compared with GFP controls. The asterisk represents significant difference as compared with control group at p
    Figure Legend Snippet: Overexpression of proNGF reduced NGF and induced expression of TrkA and p75 NTR , but not sortilin. A , B : Western blot (WB) analysis of rat retinal lysate showed significant (1.5-fold) increase in expression of TrkA and p75 NTR in rats electroporated with pGFP-proNGF123 as compared with those electroporated with pGFP (n=4). C : WB analysis showed that overexpression of pGFP-proNGF123 in rat retinas did not affect sortilin protein expression level as compared with the control rats (n=4). D : WB analysis showed that overexpression of pGFP-proNGF123 significantly reduced NGF expression (n=5) compared with GFP controls. The asterisk represents significant difference as compared with control group at p

    Techniques Used: Over Expression, Expressing, Western Blot

    10) Product Images from "NGF-trkA signaling modulates the analgesic effects of prostatic acid phosphatase in resiniferatoxin-induced neuropathy"

    Article Title: NGF-trkA signaling modulates the analgesic effects of prostatic acid phosphatase in resiniferatoxin-induced neuropathy

    Journal: Molecular Pain

    doi: 10.1177/1744806916656846

    Phenotypic changes in prostatic acid phosphatase (PAP)(+) dorsal root ganglion neurons in resiniferatoxin (RTX)-induced neuropathy. (a–e) Double immunofluorescence staining was performed with anti-activating transcription factor 3 (ATF3; a–e in red) and anti-PAP (a–e in green) antisera in the vehicle (a), RTX (b), 4-methylcatechol (4MC) (c), NGF (RTX + 2.5S NGF; d), and (e) abNGF (4MC + anti-NGF antisera) groups. (f–h) The graphs quantify the density changes in (f) PAP(+) and (g) ATF3(+) neurons and (h) the ratio changes in ATF3(+)/PAP(+) neurons according to Panels (a) to (e). (i) The mechanical thresholds were assessed using the up-and-down method with von Frey monofilaments, as described in the Materials and Methods. The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), RTX (filled bar), 4MC (grey bar), NGF (slashed bar), and abNGF (dotted bar) groups. (j, k) The graphs indicate that the mechanical thresholds correlated with PAP expression, that is, a linear correlation with (j) PAP densities and (k) an inverse correlation with the ratio of ATF3(+)/PAP(+) neurons. (l, m) The graphs show the diameter histogram of ATF3(+)/PAP(+) neurons in the RTX (l) and 4MC (M) groups. The diameter histogram shows no difference between the RTX and 4MC groups, but higher numbers of ATF3(+)/PAP(+) neurons in the RTX group. Bar, 50 µm. * p
    Figure Legend Snippet: Phenotypic changes in prostatic acid phosphatase (PAP)(+) dorsal root ganglion neurons in resiniferatoxin (RTX)-induced neuropathy. (a–e) Double immunofluorescence staining was performed with anti-activating transcription factor 3 (ATF3; a–e in red) and anti-PAP (a–e in green) antisera in the vehicle (a), RTX (b), 4-methylcatechol (4MC) (c), NGF (RTX + 2.5S NGF; d), and (e) abNGF (4MC + anti-NGF antisera) groups. (f–h) The graphs quantify the density changes in (f) PAP(+) and (g) ATF3(+) neurons and (h) the ratio changes in ATF3(+)/PAP(+) neurons according to Panels (a) to (e). (i) The mechanical thresholds were assessed using the up-and-down method with von Frey monofilaments, as described in the Materials and Methods. The graph indicates the changes in the mechanical thresholds in the vehicle (open bar), RTX (filled bar), 4MC (grey bar), NGF (slashed bar), and abNGF (dotted bar) groups. (j, k) The graphs indicate that the mechanical thresholds correlated with PAP expression, that is, a linear correlation with (j) PAP densities and (k) an inverse correlation with the ratio of ATF3(+)/PAP(+) neurons. (l, m) The graphs show the diameter histogram of ATF3(+)/PAP(+) neurons in the RTX (l) and 4MC (M) groups. The diameter histogram shows no difference between the RTX and 4MC groups, but higher numbers of ATF3(+)/PAP(+) neurons in the RTX group. Bar, 50 µm. * p

    Techniques Used: Double Immunofluorescence Staining, Expressing

    Colocalization of prostatic acid phosphatase (PAP) and high-affinity nerve growth factor (trkA) receptor after resiniferatoxin (RTX)-induced neuropathy. (a–j) Double immunofluorescence staining of dorsal root ganglia sections was performed with anti-PAP (a–e) and trkA (f-j) in the vehicle (a, f), RTX (b, g), 4-methylcatechol (4MC; c, h), (d, i) NGF (RTX + 2.5S NGF), and (e, j) abNGF (4MC + anti-NGF antisera) groups. (k, l) The graphs show the changes in (k) trkA density and (l) the colocalized ratios of PAP(+)/trkA(+) neurons in the vehicle (open bar), RTX (filled bar), 4MC (grey bar), NGF (slashed bar), and abNGF groups (dotted bar) according to Panels (a) to (j). (m) The graph shows that the mechanical thresholds correlate linearly with the ratios of PAP(+)/trkA(+) neurons. Bar, 50 µm. * p
    Figure Legend Snippet: Colocalization of prostatic acid phosphatase (PAP) and high-affinity nerve growth factor (trkA) receptor after resiniferatoxin (RTX)-induced neuropathy. (a–j) Double immunofluorescence staining of dorsal root ganglia sections was performed with anti-PAP (a–e) and trkA (f-j) in the vehicle (a, f), RTX (b, g), 4-methylcatechol (4MC; c, h), (d, i) NGF (RTX + 2.5S NGF), and (e, j) abNGF (4MC + anti-NGF antisera) groups. (k, l) The graphs show the changes in (k) trkA density and (l) the colocalized ratios of PAP(+)/trkA(+) neurons in the vehicle (open bar), RTX (filled bar), 4MC (grey bar), NGF (slashed bar), and abNGF groups (dotted bar) according to Panels (a) to (j). (m) The graph shows that the mechanical thresholds correlate linearly with the ratios of PAP(+)/trkA(+) neurons. Bar, 50 µm. * p

    Techniques Used: Double Immunofluorescence Staining

    11) Product Images from "B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS"

    Article Title: B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131780

    Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.
    Figure Legend Snippet: Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.

    Techniques Used: Expressing, Mouse Assay

    Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.
    Figure Legend Snippet: Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.

    Techniques Used: Activation Assay, Expressing, Staining, Mouse Assay, Cell Culture, In Vitro

    12) Product Images from "NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases"

    Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23020892

    NGF withdrawal for 2 days from cultured TGNs reduces CGRP release stimulated by CAP; acute NGF induces exocytosis and enhances that stimulated with low [CAP]. ( A ) Timeline for pre-treatment and experimental manipulations of TGNs to determine the effect of NGF starvation and its brief re-introduction (100 ng/mL) on CGRP release under requisite conditions. ( B ) Dose-response relationship between [CAP] and CGRP release expressed as a % of total. Asterisks show significant differences between starved and NGF acutely treated cells (* p
    Figure Legend Snippet: NGF withdrawal for 2 days from cultured TGNs reduces CGRP release stimulated by CAP; acute NGF induces exocytosis and enhances that stimulated with low [CAP]. ( A ) Timeline for pre-treatment and experimental manipulations of TGNs to determine the effect of NGF starvation and its brief re-introduction (100 ng/mL) on CGRP release under requisite conditions. ( B ) Dose-response relationship between [CAP] and CGRP release expressed as a % of total. Asterisks show significant differences between starved and NGF acutely treated cells (* p

    Techniques Used: Cell Culture

    Chimera/EA effectively inhibits 1 µM CAP-evoked CGRP release from starved TGNs, and its enhancement by NGF. TGNs were starved and incubated with 100 nM/EA using a protocol identical to that described previously for BoNT/A ( Figure 5 A). ( A ) Western blotting with anti-SNAP-25 antibodies confirms the disappearance of intact SNAP-25 and the appearance of a much faster-migrating product in cells exposed to/EA (+) but not control (−). ( B ) Histogram displaying the % of SNAP-25 cleaved, which was calculated as in Figure 5 C. ( C ) Total amounts (pg/well) of CGRP, determined as before, in control and/EA-treated cells. ( D ) Amounts of spontaneous CGRP release (% total) into HBS only, (grey bars), and that during incubation with 100 ng/mL NGF (blue bars), and ( E ) upon stimulation with 1 µM CAP (minus the spontaneous release) (mean + s.e.m. N = 3, n = 9). Unpaired one-tailed Welch test was applied to the data plotted in panels ( C – E ), * p
    Figure Legend Snippet: Chimera/EA effectively inhibits 1 µM CAP-evoked CGRP release from starved TGNs, and its enhancement by NGF. TGNs were starved and incubated with 100 nM/EA using a protocol identical to that described previously for BoNT/A ( Figure 5 A). ( A ) Western blotting with anti-SNAP-25 antibodies confirms the disappearance of intact SNAP-25 and the appearance of a much faster-migrating product in cells exposed to/EA (+) but not control (−). ( B ) Histogram displaying the % of SNAP-25 cleaved, which was calculated as in Figure 5 C. ( C ) Total amounts (pg/well) of CGRP, determined as before, in control and/EA-treated cells. ( D ) Amounts of spontaneous CGRP release (% total) into HBS only, (grey bars), and that during incubation with 100 ng/mL NGF (blue bars), and ( E ) upon stimulation with 1 µM CAP (minus the spontaneous release) (mean + s.e.m. N = 3, n = 9). Unpaired one-tailed Welch test was applied to the data plotted in panels ( C – E ), * p

    Techniques Used: Incubation, Western Blot, One-tailed Test

    BoNT/A blocked NGF-induced CGRP release and -enhancement of 20 nM CAP-evoked CGRP release. ( A ) After 2 days in the presence of 50 ng/mL NGF, TGNs were starved of the neurotrophin as detailed before, without or with the inclusion of 100 nM BoNT/A during this latter step. The release experiment was performed as described in Figure 3 A. ( B ) At the end of the protocol, one well each of BoNT/A-treated and non-treated cells were solubilised in 1× LDS and subjected to Western blotting. PVDF membranes were cut horizontally midway between the 25 k and 37 k molecular weight markers. The upper portion was exposed to antibodies reactive with syntaxin-1 (mouse monoclonal, 1:2000) and the lower piece probed with an antibody recognising both intact and BoNT/A-truncated SNAP-25 (mouse monoclonal, 1:3000). ( C ) The amount of cleaved SNAP-25 in BoNT/A-treated cells was calculated as a % (mean + s.e.m., N = 3) of the total SNAP-25 (intact + BoNT/A product). ( D ) Total CGRP (pg/well). ( E – G ) Histograms showing: ( E ) spontaneous CGRP release during the second 30 min. incubation into HBS without (grey bars) or induced by 100 ng/mL NGF (blue bars), ( F ) during the third period evoked by 20 nM CAP (minus the spontaneous release), and ( G ) during the third incubation with 1 µM CAP minus the spontaneous release. In all cases, CGRP release is expressed as a % of total CGRP (mean + s.e.m., N = 3, n = 9). Asterisks summarise the results of unpaired one-tailed Welch tests applied to the data plotted in panels E, F, and G, * p
    Figure Legend Snippet: BoNT/A blocked NGF-induced CGRP release and -enhancement of 20 nM CAP-evoked CGRP release. ( A ) After 2 days in the presence of 50 ng/mL NGF, TGNs were starved of the neurotrophin as detailed before, without or with the inclusion of 100 nM BoNT/A during this latter step. The release experiment was performed as described in Figure 3 A. ( B ) At the end of the protocol, one well each of BoNT/A-treated and non-treated cells were solubilised in 1× LDS and subjected to Western blotting. PVDF membranes were cut horizontally midway between the 25 k and 37 k molecular weight markers. The upper portion was exposed to antibodies reactive with syntaxin-1 (mouse monoclonal, 1:2000) and the lower piece probed with an antibody recognising both intact and BoNT/A-truncated SNAP-25 (mouse monoclonal, 1:3000). ( C ) The amount of cleaved SNAP-25 in BoNT/A-treated cells was calculated as a % (mean + s.e.m., N = 3) of the total SNAP-25 (intact + BoNT/A product). ( D ) Total CGRP (pg/well). ( E – G ) Histograms showing: ( E ) spontaneous CGRP release during the second 30 min. incubation into HBS without (grey bars) or induced by 100 ng/mL NGF (blue bars), ( F ) during the third period evoked by 20 nM CAP (minus the spontaneous release), and ( G ) during the third incubation with 1 µM CAP minus the spontaneous release. In all cases, CGRP release is expressed as a % of total CGRP (mean + s.e.m., N = 3, n = 9). Asterisks summarise the results of unpaired one-tailed Welch tests applied to the data plotted in panels E, F, and G, * p

    Techniques Used: Western Blot, Molecular Weight, Incubation, One-tailed Test

    Extracellular Ca 2+ is required for NGF to raise CGRP release but not for its enhancement of CAP-evoked Ca 2+ -dependent exocytosis. ( A ) NGF-starved TGNs were exposed in sequence for three 30 min. periods as follows. In the first period, all cells were exposed to HBS only. For period 2, the cells were split into three cohorts and incubated with Ca 2+ /HBS modified as follows: Cohort 1, HBS only; Cohort 2, Ca 2+ /HBS containing 100 ng/mL NGF; Cohort 3, HBS containing 100 ng/mL NGF but with Ca 2+ replaced by 2 mM EGTA. In period 3, all cells were incubated with Ca 2+ /HBS containing 20 nM CAP. ( B ) In a separate set of experiments, TGNs were processed as far as period 2 before being lysed in 1x LDS buffer, and the ERK phosphorylation was determined by Western blotting as detailed in the Materials and Methods. ( C ) Histogram showing the ratio of signal intensity for p-ERK 1/2 to total ERK 1/2 (mean + s.e.m., n ≥ 3, N = 2), determined from the requisite immuno-reactive bands detailed in panel B. ( D ) Spontaneous or NGF-induced CGRP release during incubation period 2 and ( E ) 20 nM CAP-evoked CGRP release in period 3 calculated by subtracting the amount released during incubation 1, both expressed as a % of total CGRP content; n = 9, N = 3. For all histograms, one-way ANOVA was used followed by Bonferroni’s post hoc test, and significance indicated with asterisks; * p
    Figure Legend Snippet: Extracellular Ca 2+ is required for NGF to raise CGRP release but not for its enhancement of CAP-evoked Ca 2+ -dependent exocytosis. ( A ) NGF-starved TGNs were exposed in sequence for three 30 min. periods as follows. In the first period, all cells were exposed to HBS only. For period 2, the cells were split into three cohorts and incubated with Ca 2+ /HBS modified as follows: Cohort 1, HBS only; Cohort 2, Ca 2+ /HBS containing 100 ng/mL NGF; Cohort 3, HBS containing 100 ng/mL NGF but with Ca 2+ replaced by 2 mM EGTA. In period 3, all cells were incubated with Ca 2+ /HBS containing 20 nM CAP. ( B ) In a separate set of experiments, TGNs were processed as far as period 2 before being lysed in 1x LDS buffer, and the ERK phosphorylation was determined by Western blotting as detailed in the Materials and Methods. ( C ) Histogram showing the ratio of signal intensity for p-ERK 1/2 to total ERK 1/2 (mean + s.e.m., n ≥ 3, N = 2), determined from the requisite immuno-reactive bands detailed in panel B. ( D ) Spontaneous or NGF-induced CGRP release during incubation period 2 and ( E ) 20 nM CAP-evoked CGRP release in period 3 calculated by subtracting the amount released during incubation 1, both expressed as a % of total CGRP content; n = 9, N = 3. For all histograms, one-way ANOVA was used followed by Bonferroni’s post hoc test, and significance indicated with asterisks; * p

    Techniques Used: Sequencing, Incubation, Modification, Western Blot

    NGF induces a minor increase in Ca 2+ - and SNARE-dependent CGRP release, whereas it greatly enhances the CAP-evoked exocytosis which is blocked by BoNT/A and/EA at low [CAP] but only abolished by BoNT/EA at higher [CAP]. ( A ) Illustrates the effect of acute NGF on CGRP exocytosis from control neonatal rat TGNs starved of the neurotrophin for 2 days, and ( B ) in TGNs pre-treated with BoNT/A or /EA. ( A ) NGF binds to its receptor TrkA, activates the signalling cascades shown [ 18 ], and induces Ca 2+ influx by an unidentified mechanism ( ? ). Elevated intracellular Ca 2+ ([Ca 2+ ] i ) triggers the fusion of large dense core vesicles (LDCVs) via SNARE-complexes (VAMP, syntaxin-1 and SNAP-25), thereby, causing exocytotic release of CGRP and surface delivery of vesicle constituents. This acute potentiation by NGF can involve the phosphatidylinositol 3-kinase—Src ( PI3K-Src) pathway, which promotes trafficking of LDCVs, and insertion of their TRPV1 channels into the plasmalemma by Ca 2+ -regulated exocytosis (blue arrows) c.f. [ 18 , 21 ]. Additionally, the phospholipase C γ (PLCγ) cascade leads to sensitisation of TRPV1 already on the plasmalemma (red dashed arrows) [ 18 ]. The outcome of these composite influences of NGF on TRPV1 is that when the channel is activated by CAP [Ca 2+ ] I is raised even more than normally [ 15 , 18 , 21 ] and this further enhances CGRP release ( Figure 2 ). ( B ) The proteases of BoNT/A and/EA delete 9 (purple arrow) and 26 (green arrow) residues from SNAP-25 (Insert), respectively, preventing the fusion of LDCVs; this blocks the minimal CGRP exocytosis elicited by NGF (arrow with crosses, left) and its enhancement of the release evoked by 20 nM CAP ( ) (arrow with crosses, centre). Stronger stimulation of TRPV1 with 1 µM CAP ( ) induces a lot more Ca 2+ influx ( [Ca 2+ ] i , ( [Ca 2+ ] i , ( [Ca 2+ ] i ; Figure 1 ) which causes a moderate increase in CGRP release but overcomes the inhibition by BoNT/A (purple cross with broken lines, right) while/EA (green cross, right) remains effective in diminishing CGRP release. Acute sensitisation by NGF of TRPV1 selectively enhances neuropeptide exocytosis stimulated by low [CAP] (
    Figure Legend Snippet: NGF induces a minor increase in Ca 2+ - and SNARE-dependent CGRP release, whereas it greatly enhances the CAP-evoked exocytosis which is blocked by BoNT/A and/EA at low [CAP] but only abolished by BoNT/EA at higher [CAP]. ( A ) Illustrates the effect of acute NGF on CGRP exocytosis from control neonatal rat TGNs starved of the neurotrophin for 2 days, and ( B ) in TGNs pre-treated with BoNT/A or /EA. ( A ) NGF binds to its receptor TrkA, activates the signalling cascades shown [ 18 ], and induces Ca 2+ influx by an unidentified mechanism ( ? ). Elevated intracellular Ca 2+ ([Ca 2+ ] i ) triggers the fusion of large dense core vesicles (LDCVs) via SNARE-complexes (VAMP, syntaxin-1 and SNAP-25), thereby, causing exocytotic release of CGRP and surface delivery of vesicle constituents. This acute potentiation by NGF can involve the phosphatidylinositol 3-kinase—Src ( PI3K-Src) pathway, which promotes trafficking of LDCVs, and insertion of their TRPV1 channels into the plasmalemma by Ca 2+ -regulated exocytosis (blue arrows) c.f. [ 18 , 21 ]. Additionally, the phospholipase C γ (PLCγ) cascade leads to sensitisation of TRPV1 already on the plasmalemma (red dashed arrows) [ 18 ]. The outcome of these composite influences of NGF on TRPV1 is that when the channel is activated by CAP [Ca 2+ ] I is raised even more than normally [ 15 , 18 , 21 ] and this further enhances CGRP release ( Figure 2 ). ( B ) The proteases of BoNT/A and/EA delete 9 (purple arrow) and 26 (green arrow) residues from SNAP-25 (Insert), respectively, preventing the fusion of LDCVs; this blocks the minimal CGRP exocytosis elicited by NGF (arrow with crosses, left) and its enhancement of the release evoked by 20 nM CAP ( ) (arrow with crosses, centre). Stronger stimulation of TRPV1 with 1 µM CAP ( ) induces a lot more Ca 2+ influx ( [Ca 2+ ] i , ( [Ca 2+ ] i , ( [Ca 2+ ] i ; Figure 1 ) which causes a moderate increase in CGRP release but overcomes the inhibition by BoNT/A (purple cross with broken lines, right) while/EA (green cross, right) remains effective in diminishing CGRP release. Acute sensitisation by NGF of TRPV1 selectively enhances neuropeptide exocytosis stimulated by low [CAP] (

    Techniques Used: Inhibition

    Capsaicin (CAP) induces dose-dependent increases of [Ca 2+ ] i in cultured trigeminal ganglion neurons (TGNs). Neurons were cultured for 4 days with nerve growth factor (NGF) before being loaded with Fluo-4 AM and recording fluorescence intensity by confocal microscopy. ( A ) Solid lines indicate the mean fluorescence (F) relative to initial fluorescence (F 0 ), which was calculated [(F − F 0 )/F 0 ] for cells exposed to different concentrations of CAP ([CAP]) and plotted against time. Broken lines indicate mean values +s.e.m. (standard error of the mean) n > 50 cells for each [CAP]. ( B ) Area under the curve (AUC) of mean fluorescence traces recorded for each [CAP] exposure. Column heights and error bars indicate mean AUC and s.e.m, respectively.
    Figure Legend Snippet: Capsaicin (CAP) induces dose-dependent increases of [Ca 2+ ] i in cultured trigeminal ganglion neurons (TGNs). Neurons were cultured for 4 days with nerve growth factor (NGF) before being loaded with Fluo-4 AM and recording fluorescence intensity by confocal microscopy. ( A ) Solid lines indicate the mean fluorescence (F) relative to initial fluorescence (F 0 ), which was calculated [(F − F 0 )/F 0 ] for cells exposed to different concentrations of CAP ([CAP]) and plotted against time. Broken lines indicate mean values +s.e.m. (standard error of the mean) n > 50 cells for each [CAP]. ( B ) Area under the curve (AUC) of mean fluorescence traces recorded for each [CAP] exposure. Column heights and error bars indicate mean AUC and s.e.m, respectively.

    Techniques Used: Cell Culture, Fluorescence, Confocal Microscopy

    Deprival of NGF for 2 days does not significantly alter total protein or calcitonin gene-related peptide (CGRP) contents of rat cultured TGNs but reduces both spontaneous and CAP-evoked exocytosis of the neuropeptide. ( A ) Schematic illustrating the experimental protocol. Rat TGNs were cultivated initially in the presence of NGF (50 ng/mL) for 2 days before its withdrawal from half of the wells (starved) or retention for the other cohort over the 4 days (fed). The amounts of CGRP released were quantified during sequential 30 min. exposures, firstly to HEPES buffered saline (HBS) only (( D ), spontaneous release), and then to 20 nM CAP in HBS ( E ). At the end of experiments, cells were solubilised with 1% ( v / v ) Triton X-100 in HBS, then total protein ( B ) and CGRP contents ( C ) were determined, as detailed in Materials and Methods. Data are presented as mean + s.e.m., n ≥ 12, N = 3. Asterisks summarise the results of unpaired t -tests with Welch’s correction, ** p
    Figure Legend Snippet: Deprival of NGF for 2 days does not significantly alter total protein or calcitonin gene-related peptide (CGRP) contents of rat cultured TGNs but reduces both spontaneous and CAP-evoked exocytosis of the neuropeptide. ( A ) Schematic illustrating the experimental protocol. Rat TGNs were cultivated initially in the presence of NGF (50 ng/mL) for 2 days before its withdrawal from half of the wells (starved) or retention for the other cohort over the 4 days (fed). The amounts of CGRP released were quantified during sequential 30 min. exposures, firstly to HEPES buffered saline (HBS) only (( D ), spontaneous release), and then to 20 nM CAP in HBS ( E ). At the end of experiments, cells were solubilised with 1% ( v / v ) Triton X-100 in HBS, then total protein ( B ) and CGRP contents ( C ) were determined, as detailed in Materials and Methods. Data are presented as mean + s.e.m., n ≥ 12, N = 3. Asterisks summarise the results of unpaired t -tests with Welch’s correction, ** p

    Techniques Used: Cell Culture

    13) Product Images from "Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation"

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    Journal: Journal of Radiation Research

    doi: 10.1093/jrr/rrx032

    Hypothesis of inhibition mechanism of the NGF-induced neurite extension by chronic irradiation with low-dose-rate 137 Cs-γ rays via Ca 2+ /calmodulin-dependent kinase II activation. Chronic irradiation with low-dose-rate γ rays activates CaMKII in the downstream of the Ca-dependent signal induced by NGF. The production of reactive oxygen species generated by irradiation may be involved in this activation of CaMKII. The CaMK II suppresses NGF-induced neuronal extension by inhibition of the activation of Rac1 downstream of the PI3K-Akt signal. IQGAP1, which is considered to contribute to neurite extension, may be involved in this inhibition of Rac1 activity. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase, PI3K = phosphoinositide 3-kinase, IP3 = inositol trisphosphate, CaM = calmodulin, CaMKII = Ca 2+ /calmodulin-dependent protein kinase II, ROS = reactive oxygen species, IQGAP1 = Ras GTPase-activating-like protein.
    Figure Legend Snippet: Hypothesis of inhibition mechanism of the NGF-induced neurite extension by chronic irradiation with low-dose-rate 137 Cs-γ rays via Ca 2+ /calmodulin-dependent kinase II activation. Chronic irradiation with low-dose-rate γ rays activates CaMKII in the downstream of the Ca-dependent signal induced by NGF. The production of reactive oxygen species generated by irradiation may be involved in this activation of CaMKII. The CaMK II suppresses NGF-induced neuronal extension by inhibition of the activation of Rac1 downstream of the PI3K-Akt signal. IQGAP1, which is considered to contribute to neurite extension, may be involved in this inhibition of Rac1 activity. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase, PI3K = phosphoinositide 3-kinase, IP3 = inositol trisphosphate, CaM = calmodulin, CaMKII = Ca 2+ /calmodulin-dependent protein kinase II, ROS = reactive oxygen species, IQGAP1 = Ras GTPase-activating-like protein.

    Techniques Used: Inhibition, Irradiation, Activation Assay, Generated, Activity Assay, Chick Chorioallantoic Membrane Assay

    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Irradiation

    Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.
    Figure Legend Snippet: Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Techniques Used: Western Blot, Irradiation

    Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Inhibition, Activity Assay, Irradiation

    Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Techniques Used: Irradiation, Activation Assay, Activity Assay

    14) Product Images from "Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development"

    Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129944

    Distinct Kidins220 splice isoforms display specific cellular localisations. (A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.
    Figure Legend Snippet: Distinct Kidins220 splice isoforms display specific cellular localisations. (A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Variant Assay

    15) Product Images from "Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors"

    Article Title: Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors

    Journal: iScience

    doi: 10.1016/j.isci.2022.104348

    Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.
    Figure Legend Snippet: Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.

    Techniques Used: Functional Assay, Sequencing, Binding Assay, Construct, Western Blot, Transfection, Concentration Assay, Labeling, Incubation

    TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.
    Figure Legend Snippet: TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.

    Techniques Used: Functional Assay, Binding Assay, Construct, Transfection

    Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.
    Figure Legend Snippet: Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.

    Techniques Used: Nuclear Magnetic Resonance

    16) Product Images from "A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers"

    Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

    Journal: Autophagy

    doi: 10.1080/15548627.2020.1794590

    SMK-17 induces autophagy through TFEB activation. (A) Representative images and (B) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 1 h. ( C, D, E ) SMK-17 induces expression of TFEB target genes. (C) NGF-differentiated PC12D cells were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (D) Knockdown of TFEB in NGF-differentiated PC12D cells was confirmed with western blotting. (E) NGF-differentiated PC12D cells transfected with Tfeb siRNA or control siRNA were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (F) Involvement of TFEB in SMK-17-induced autophagy. NGF-differentiated PC12D cells expressing GFP-LC3-RFP were transfected with Tfeb siRNA or control siRNA and then treated with 10 µM SMK-17 for 24 h. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. Scale bar: 20 µm. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p
    Figure Legend Snippet: SMK-17 induces autophagy through TFEB activation. (A) Representative images and (B) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 1 h. ( C, D, E ) SMK-17 induces expression of TFEB target genes. (C) NGF-differentiated PC12D cells were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (D) Knockdown of TFEB in NGF-differentiated PC12D cells was confirmed with western blotting. (E) NGF-differentiated PC12D cells transfected with Tfeb siRNA or control siRNA were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (F) Involvement of TFEB in SMK-17-induced autophagy. NGF-differentiated PC12D cells expressing GFP-LC3-RFP were transfected with Tfeb siRNA or control siRNA and then treated with 10 µM SMK-17 for 24 h. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. Scale bar: 20 µm. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p

    Techniques Used: Activation Assay, Nuclear Translocation Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Western Blot, Transfection

    Memantine and clemastine induce ER stress. (A) Effect of memantine, clemastine, and flunarizine on the expression of ER stress markers. NGF-differentiated PC12D cells were treated with 2 µM tunicamycin (Tm), 10 mM 2-deoxyglucose (2-DG), 100 µM memantine (Mem), 5 µM clemastine (Cle), or 20 µM flunarizine (Flu). After 12 h (for detection of EIF2S1 phosphorylation) or 24 h (for detection of HSPA5 and DDIT3 expression), the cells were collected and subjected to western blotting analysis with the indicated antibodies. (B) Memantine and clemastine induce EIF2AK3 phosphorylation. NGF-differentiated PC12D cells were treated with the indicated compounds at the same concentrations as described in (A). After 12 h, the cells were collected and subjected to western blotting analysis with the indicated antibodies. (C) Memantine and clemastine induce alternative Xbp1 mRNA splicing. NGF-differentiated PC12D cells were treated with the indicated compounds for 12 h at the same concentrations as described in (A). Unspliced (U) and spliced (S) Xbp1 were detected by RT-PCR. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p
    Figure Legend Snippet: Memantine and clemastine induce ER stress. (A) Effect of memantine, clemastine, and flunarizine on the expression of ER stress markers. NGF-differentiated PC12D cells were treated with 2 µM tunicamycin (Tm), 10 mM 2-deoxyglucose (2-DG), 100 µM memantine (Mem), 5 µM clemastine (Cle), or 20 µM flunarizine (Flu). After 12 h (for detection of EIF2S1 phosphorylation) or 24 h (for detection of HSPA5 and DDIT3 expression), the cells were collected and subjected to western blotting analysis with the indicated antibodies. (B) Memantine and clemastine induce EIF2AK3 phosphorylation. NGF-differentiated PC12D cells were treated with the indicated compounds at the same concentrations as described in (A). After 12 h, the cells were collected and subjected to western blotting analysis with the indicated antibodies. (C) Memantine and clemastine induce alternative Xbp1 mRNA splicing. NGF-differentiated PC12D cells were treated with the indicated compounds for 12 h at the same concentrations as described in (A). Unspliced (U) and spliced (S) Xbp1 were detected by RT-PCR. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    SMK-17 induces PRKC/PKC-dependent TFEB activation and clearance of intracellular aggregates. (A, B) Western blotting analyses of NGF-differentiated PC12D cells treated with (A) 10 µM SMK-17 or 100 nM phorbol 12-myristate 13-acetate (PMA) for the indicated times, or (B) 10 µM SMK-17 in the presence or absence of 5 µM PRKC inhibitor (PRKCi, Gö6983) for 3 h. Phosphorylation of PRKC substrates was detected by using p-(Ser) PRKC substrate antibody. (C) Representative images and (D) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1 or 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Scale bar: 20 µm. (E) Representative images and (F) quantification of LysoTracker Red DND-99 staining assay results. NGF-differentiated PC12D cells were treated with 100 nM torin1, 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Mean fluorescent intensity was quantified. Scale bar: 20 µm. (G) NGF-differentiated PC12D cells expressing GFP-LC3-RFP were treated with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. (H) Representative images and (I) quantification of aggresome clearance assay results. NGF-differentiated PC12D cells were treated with MPP + for 16 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 8 h in the presence or absence of 5 µM PRKCi. The number of aggresoe dots per cell in each image was quantified. Scale bar: 20 µm. (J) Western blotting analysis of NGF-differentiated PC12D cells transiently transfected with GFP, GFP-HTTQ23, or GFP-HTTQ74 for 72 h. (K) Representative images and (L) quantification of mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p
    Figure Legend Snippet: SMK-17 induces PRKC/PKC-dependent TFEB activation and clearance of intracellular aggregates. (A, B) Western blotting analyses of NGF-differentiated PC12D cells treated with (A) 10 µM SMK-17 or 100 nM phorbol 12-myristate 13-acetate (PMA) for the indicated times, or (B) 10 µM SMK-17 in the presence or absence of 5 µM PRKC inhibitor (PRKCi, Gö6983) for 3 h. Phosphorylation of PRKC substrates was detected by using p-(Ser) PRKC substrate antibody. (C) Representative images and (D) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1 or 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Scale bar: 20 µm. (E) Representative images and (F) quantification of LysoTracker Red DND-99 staining assay results. NGF-differentiated PC12D cells were treated with 100 nM torin1, 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Mean fluorescent intensity was quantified. Scale bar: 20 µm. (G) NGF-differentiated PC12D cells expressing GFP-LC3-RFP were treated with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. (H) Representative images and (I) quantification of aggresome clearance assay results. NGF-differentiated PC12D cells were treated with MPP + for 16 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 8 h in the presence or absence of 5 µM PRKCi. The number of aggresoe dots per cell in each image was quantified. Scale bar: 20 µm. (J) Western blotting analysis of NGF-differentiated PC12D cells transiently transfected with GFP, GFP-HTTQ23, or GFP-HTTQ74 for 72 h. (K) Representative images and (L) quantification of mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p

    Techniques Used: Activation Assay, Western Blot, Nuclear Translocation Assay, Stable Transfection, Expressing, Staining, Transfection, Mutagenesis

    Activities of autophagy inducers in inhibition of aggresome formation and clearance of protein aggregates. (A) Representative images and (B) quantification of aggresome formation assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 24 h in the presence or absence of 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (C) Representative images and (D) quantification of aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH for 8 h. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (E) Quantification of the aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with the indicated compounds for 8 h. See also Table 1 and Fig. S3A . The number of aggresome dots per cell in each image was quantified. (F) Quantification of the mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with the indicated compounds for 24 h. See also Table 1 and Fig. S3B . Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. (G) Cytotoxicity of the autophagy inducers against primary cultured rat cortical neurons. Cells were treated with the indicated compounds for 24 h, and cytotoxicity was measured by LDH release assay. The data are expressed as a percentage of total amount of LDH analyzed in each plate. Data are shown as mean ± SD (n = 3 [ B, D, E, F ], n = 5 [ G ]). ### p
    Figure Legend Snippet: Activities of autophagy inducers in inhibition of aggresome formation and clearance of protein aggregates. (A) Representative images and (B) quantification of aggresome formation assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 24 h in the presence or absence of 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (C) Representative images and (D) quantification of aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH for 8 h. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (E) Quantification of the aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with the indicated compounds for 8 h. See also Table 1 and Fig. S3A . The number of aggresome dots per cell in each image was quantified. (F) Quantification of the mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with the indicated compounds for 24 h. See also Table 1 and Fig. S3B . Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. (G) Cytotoxicity of the autophagy inducers against primary cultured rat cortical neurons. Cells were treated with the indicated compounds for 24 h, and cytotoxicity was measured by LDH release assay. The data are expressed as a percentage of total amount of LDH analyzed in each plate. Data are shown as mean ± SD (n = 3 [ B, D, E, F ], n = 5 [ G ]). ### p

    Techniques Used: Inhibition, Tube Formation Assay, Mutagenesis, Transfection, Cell Culture, Lactate Dehydrogenase Assay

    SMK-17 induces autophagy in a MAP2 K inhibition- or MTOR-independent manner. (A) Chemical structure of SMK-17. (B, C) Western blotting analyses of NGF-differentiated PC12D cells treated with (B) 10 µM SMK-17 for the indicated times, (C) 10 µM SMK-17 in the presence or absence of 100 nM bafilomycin A 1 (Baf A 1 ) for 24 h with the indicated antibodies. (D) NGF-differentiated PC12D cells transfected with the mCherry-GFP-LC3 (tfLC3) plasmid vector were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 8 h. Autophagy flux was observed under confocal microscopy. Scale bar: 20 µm. (E, F) Western blotting analyses of NGF-differentiated PC12D cells treated with (E) 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 4 h, or (F) 100 nM torin1, 10 µM SMK-17, or 10 µM U0126 for 1 h with the indicated antibodies
    Figure Legend Snippet: SMK-17 induces autophagy in a MAP2 K inhibition- or MTOR-independent manner. (A) Chemical structure of SMK-17. (B, C) Western blotting analyses of NGF-differentiated PC12D cells treated with (B) 10 µM SMK-17 for the indicated times, (C) 10 µM SMK-17 in the presence or absence of 100 nM bafilomycin A 1 (Baf A 1 ) for 24 h with the indicated antibodies. (D) NGF-differentiated PC12D cells transfected with the mCherry-GFP-LC3 (tfLC3) plasmid vector were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 8 h. Autophagy flux was observed under confocal microscopy. Scale bar: 20 µm. (E, F) Western blotting analyses of NGF-differentiated PC12D cells treated with (E) 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 4 h, or (F) 100 nM torin1, 10 µM SMK-17, or 10 µM U0126 for 1 h with the indicated antibodies

    Techniques Used: Inhibition, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy

    17) Product Images from "Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors"

    Article Title: Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors

    Journal: iScience

    doi: 10.1016/j.isci.2022.104348

    Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.
    Figure Legend Snippet: Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.

    Techniques Used: Functional Assay, Sequencing, Binding Assay, Construct, Western Blot, Transfection, Concentration Assay, Labeling, Incubation

    TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.
    Figure Legend Snippet: TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.

    Techniques Used: Functional Assay, Binding Assay, Construct, Transfection

    Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.
    Figure Legend Snippet: Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.

    Techniques Used: Nuclear Magnetic Resonance

    18) Product Images from "L1CAM and its cell-surface mutants: new mechanisms and effects relevant to the physiology and pathology of neural cells"

    Article Title: L1CAM and its cell-surface mutants: new mechanisms and effects relevant to the physiology and pathology of neural cells

    Journal: Journal of Neurochemistry

    doi: 10.1111/jnc.12015

    Neurite outgrowth induced by treatment with Y27632 (a) and nerve growth factor (NGF) (b–e) in PC12-27 and PC12-TrkA cells non-transfected and transfected with various forms of L1 cell adhesion molecule (L1CAM). A: Immunofluorescence (red = L1CAM; green = β-tubulin; blue = DAPI) of PC12-27 cells stably transfected with wt and mutated L1CAMs, treated for 60 min with Y27632 (25 μM). The images shown are representative of at least 18 images from three experiments. The bar on the right, valid for all A panels, is of 20 μm. (b) Immunofluorescence as in (a), however, of PC12-27 cells transiently transfected with wtL1CAM and mutants, treated with NGF (100 ng/mL) for 48 h. Quantization of the results in (d) (histograms are averages of > 150 cells from three experiments ± SD). (c) Immunofluorescence as in (a), however, of PC12-TrkA cells transiently transfected with wtL1CAM and mutants, treated with NGF as in (b). Quantization of the results in (e) (histograms are averages of > 150 cells from three experiments ± SD). The bar in panel (c) right, valid for all panels of (b and c), is of 20 μm. Significance of quantitative changes observed with respect to non-transfected PC12-27 and PC12-TrkA [left columns in (d) and (e)] is indicated as in Fig. 2 a.
    Figure Legend Snippet: Neurite outgrowth induced by treatment with Y27632 (a) and nerve growth factor (NGF) (b–e) in PC12-27 and PC12-TrkA cells non-transfected and transfected with various forms of L1 cell adhesion molecule (L1CAM). A: Immunofluorescence (red = L1CAM; green = β-tubulin; blue = DAPI) of PC12-27 cells stably transfected with wt and mutated L1CAMs, treated for 60 min with Y27632 (25 μM). The images shown are representative of at least 18 images from three experiments. The bar on the right, valid for all A panels, is of 20 μm. (b) Immunofluorescence as in (a), however, of PC12-27 cells transiently transfected with wtL1CAM and mutants, treated with NGF (100 ng/mL) for 48 h. Quantization of the results in (d) (histograms are averages of > 150 cells from three experiments ± SD). (c) Immunofluorescence as in (a), however, of PC12-TrkA cells transiently transfected with wtL1CAM and mutants, treated with NGF as in (b). Quantization of the results in (e) (histograms are averages of > 150 cells from three experiments ± SD). The bar in panel (c) right, valid for all panels of (b and c), is of 20 μm. Significance of quantitative changes observed with respect to non-transfected PC12-27 and PC12-TrkA [left columns in (d) and (e)] is indicated as in Fig. 2 a.

    Techniques Used: Transfection, Immunofluorescence, Stable Transfection

    Dose-dependent neurite outgrowth induced by nerve growth factor (NGF) and/or recombinant, water-soluble chimera including the ectodomain of L1CAM and the Fc domain of human IgGs (L1CAM-Fc) in PC12-TrkA cells. Effects of inhibitory drugs. (a) The immunofluorescence of β-tubulin [green; nuclei labeled blue by 4'-6-Diamidino-2-phenylindole (DAPI)] illustrates the phenotype of PC12-TrkA cells treated for 24 h with increasing concentrations of L1CAM-Fc alone (upper row) or together with a small concentration (5 ng/mL) of NGF (lower row), as specified by the white numbers that appear over the panels. The bar on the bottom right, valid for all (a) panels, is of 20 μm. (b) Left and right, quantization of the results taken from the data illustrated in (a), upper and lower rows: average neurite lengths ± SD ( > 150 cells per sample from three experiments/histogram; significance calculated with respect to the data without L1CAM-Fc- marked as in Fig. 2 a); and % cells exhibiting thin neurites, > 10 μm long, specified by the numbers written in red over the histograms. (c) Effects on neurite outgrowth and on the % cells with thin neurites [marked as in (b)] induced in PC12-TrkA cells by the NGF-binding drug Y1036 (left), the TrkA inhibitor Calbiochem 648450 (Trk-I, middle), and the p75 NTR peptide inhibitor TAT-Pep5 (p75 NTR -I, right), administered at the indicated concentrations 1 h before NGF and/or L1CAM-Fc application, and then maintained together with the latter for 24 h. To evaluate the effects of the drugs, compare the individual data in (c) with the corresponding data with and without L1CAM-Fc shown in (b).
    Figure Legend Snippet: Dose-dependent neurite outgrowth induced by nerve growth factor (NGF) and/or recombinant, water-soluble chimera including the ectodomain of L1CAM and the Fc domain of human IgGs (L1CAM-Fc) in PC12-TrkA cells. Effects of inhibitory drugs. (a) The immunofluorescence of β-tubulin [green; nuclei labeled blue by 4'-6-Diamidino-2-phenylindole (DAPI)] illustrates the phenotype of PC12-TrkA cells treated for 24 h with increasing concentrations of L1CAM-Fc alone (upper row) or together with a small concentration (5 ng/mL) of NGF (lower row), as specified by the white numbers that appear over the panels. The bar on the bottom right, valid for all (a) panels, is of 20 μm. (b) Left and right, quantization of the results taken from the data illustrated in (a), upper and lower rows: average neurite lengths ± SD ( > 150 cells per sample from three experiments/histogram; significance calculated with respect to the data without L1CAM-Fc- marked as in Fig. 2 a); and % cells exhibiting thin neurites, > 10 μm long, specified by the numbers written in red over the histograms. (c) Effects on neurite outgrowth and on the % cells with thin neurites [marked as in (b)] induced in PC12-TrkA cells by the NGF-binding drug Y1036 (left), the TrkA inhibitor Calbiochem 648450 (Trk-I, middle), and the p75 NTR peptide inhibitor TAT-Pep5 (p75 NTR -I, right), administered at the indicated concentrations 1 h before NGF and/or L1CAM-Fc application, and then maintained together with the latter for 24 h. To evaluate the effects of the drugs, compare the individual data in (c) with the corresponding data with and without L1CAM-Fc shown in (b).

    Techniques Used: Recombinant, Immunofluorescence, Labeling, Concentration Assay, Binding Assay

    Phosphorylation of the TrkA receptor at the Y490 site induced by nerve growth factor (NGF) and recombinant, water-soluble chimera including the ectodomain of L1CAM and the Fc domain of human IgGs (L1CAM-Fc) in canonical PC12, PC12-27, and PC12-TrkA cells. Parallel aliquots of canonical PC12 cells (a), PC12-27 (b), and PC12-TrkA cells (c) were exposed for 20 min to NGF (50 ng/mL), L1CAM-Fc (500 ng/mL), or the two together. Y490 phosphorylation of TrkA was analyzed in western blots stained with the specific anti-pTrkA Ab. The values shown were calculated with respect to the constitutive phosphorylation level of TrkA (basal), which in PC12-27 cells was 10% and in PC12-TrkA was 300% with respect to the canonical PC12. In each panel, the significance of the differences (with respect to the basal phosphorylation levels) is marked as in Fig. 2 a.
    Figure Legend Snippet: Phosphorylation of the TrkA receptor at the Y490 site induced by nerve growth factor (NGF) and recombinant, water-soluble chimera including the ectodomain of L1CAM and the Fc domain of human IgGs (L1CAM-Fc) in canonical PC12, PC12-27, and PC12-TrkA cells. Parallel aliquots of canonical PC12 cells (a), PC12-27 (b), and PC12-TrkA cells (c) were exposed for 20 min to NGF (50 ng/mL), L1CAM-Fc (500 ng/mL), or the two together. Y490 phosphorylation of TrkA was analyzed in western blots stained with the specific anti-pTrkA Ab. The values shown were calculated with respect to the constitutive phosphorylation level of TrkA (basal), which in PC12-27 cells was 10% and in PC12-TrkA was 300% with respect to the canonical PC12. In each panel, the significance of the differences (with respect to the basal phosphorylation levels) is marked as in Fig. 2 a.

    Techniques Used: Recombinant, Western Blot, Staining

    19) Product Images from "Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration"

    Article Title: Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration

    Journal: ACS chemical neuroscience

    doi: 10.1021/acschemneuro.1c00854

    HJ-01and HJ-02 reduce the interaction between PTPσ and TrkA. Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p
    Figure Legend Snippet: HJ-01and HJ-02 reduce the interaction between PTPσ and TrkA. Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p

    Techniques Used: Western Blot, Immunoprecipitation, Transfection

    20) Product Images from "Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors"

    Article Title: Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors

    Journal: iScience

    doi: 10.1016/j.isci.2022.104348

    Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.
    Figure Legend Snippet: Functional assays of TrkA-eJTM-P/G mutants (A) Protein sequence alignment of the eJTM region of TrkA between different species: human, rat, mouse, and chicken. The position of the Pro residues studied is shown in bold. Below the protein sequence are shown the locations of 2P, 3P, and 5P discussed in the text. (B) Binding assays of biotinylated NGF to the indicated constructs of TrkA expressed in HEK293 cells and fitted to one-site binding. Paired t test showed significant differences between TrkA-wt and TrkA-5P/5G with a p = 0.036. (C) Western blot and quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with increasing concentration of NGF (0, 0.1, 1, 10, and 100 ng/mL) for 15 min. Data are represented as mean ± SEM. Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by ordinary one-way ANOVA analysis with Dunnett’s multiple comparison test. Exact p values are shown. ns, not significant. (D) FITC-labeled HEK293 cells transfected with the indicated TrkA constructs incubated with a FITC-labeled antibody against the N-terminal region of TrkA show that all constructs are equally expressed at the plasma membrane.

    Techniques Used: Functional Assay, Sequencing, Binding Assay, Construct, Western Blot, Transfection, Concentration Assay, Labeling, Incubation

    TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.
    Figure Legend Snippet: TrkA domains and functional assays of TrkA-ΔeJTM (A) A drawing showing the location of the protein domains is shown on the left of an intrinsic order prediction using IUPred2A ( https://iupred2a.elte.hu/ ) plotted versus the residue number of TrkA. A value above 0.5 indicates disorder. The eJTM region corresponding to residues 383–414 is shown in green. The residues previously shown to have an effect of NGF binding to TrkA when mutated are shown in red bold. The proline residues present in this region are shown in black bold. Residues Lys 410 and 411 are shown in blue bold. (B) Crystal structure of TrkA-d5 domain in complex with NGF (PDB: 1www ) indicating the residue P349, which is the last residue of TrKA construct observed in the X-ray structure. (C) Binding assays of biotinylated NGF to TrkA and TrkA-ΔeJTM expressed in HEK293 cells and fitted to one-site binding. (D) Quantification using TrkA phospho-specific antibodies of cell lysate extracts from HEK293 cells transfected with the indicated constructs and stimulated with NGF for the indicated times (0, 5, and 15 min). (E) Percentage of differentiated PC12nnr5 cells with NGF transfected with the indicated constructs stimulated with NGF for 48 h. Data are represented as mean ± SEM Error bars represent the SEM of at least three independent experiments. Statistical significance obtained by two-way ANOVA analysis with Bonferroni correction. Exact p values are shown.

    Techniques Used: Functional Assay, Binding Assay, Construct, Transfection

    Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.
    Figure Legend Snippet: Conservation of the eJTM region in Trk recepotrs (A) Alignment of eJTM regions of three human Trk receptors. Proline residues are in bold; hydrophobic and aromatic residues are shown in orange. (B) Superposition of crystal structures solved for the complex of TrkA-d5 domain with NGF (cyan, PDB: 1WWW ) and the complex of TrkB-d5 domain with BDNF (orange, PDB: 1HCF ). (C) Schematic representation of the NGF/TrkA complex based on the crystal structure of the NGF/TrkA-d5 complex (PDB: 1WWW ) and the NMR structure of TrkA-TMD dimer in DPC micelles (PDB: 2N90 ). d5, TrkA-d5 domain of TrkA; eJTM, extracellular juxtamembrane region of TrkA; TMD, transmembrane domain of TrkA. The location of Lys410 is indicated.

    Techniques Used: Nuclear Magnetic Resonance

    21) Product Images from "Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells by Ifenprodil: The Role of Sigma-1 and IP3 Receptors"

    Article Title: Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells by Ifenprodil: The Role of Sigma-1 and IP3 Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037989

    Effects of IP 3 receptor antagonists on NGF-induced neurite outgrowth in PC12 cells. (A): In the presence of NGF (2.5 ng/ml), vehicle, ifenprodil (10 µM), ifenprodil (10 µM)+xestospongin C (1.0 µM), xestospongin C (1.0 µM) were incubated with PC12 cells. (B): In the presence of NGF (2.5 ng/ml), vehicle, ifenprodil (10 µM), ifenprodil (10 µM)+2-APB (100 µM), or 2-APB (100 µM) were incubated in PC12 cells. Four days after incubation with test drugs, morphometric analysis was performed. The data show the mean ± SEM (n = 6). ***p
    Figure Legend Snippet: Effects of IP 3 receptor antagonists on NGF-induced neurite outgrowth in PC12 cells. (A): In the presence of NGF (2.5 ng/ml), vehicle, ifenprodil (10 µM), ifenprodil (10 µM)+xestospongin C (1.0 µM), xestospongin C (1.0 µM) were incubated with PC12 cells. (B): In the presence of NGF (2.5 ng/ml), vehicle, ifenprodil (10 µM), ifenprodil (10 µM)+2-APB (100 µM), or 2-APB (100 µM) were incubated in PC12 cells. Four days after incubation with test drugs, morphometric analysis was performed. The data show the mean ± SEM (n = 6). ***p

    Techniques Used: Incubation

    22) Product Images from "A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria"

    Article Title: A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201705085

    Foretinib and lestaurtinib inhibit the low levels of TrkA tyrosine phosphorylation in NGF-deprived neurons and suppress axon degeneration. (A) Quantitative normalized representation of the phosphoproteomics analysis performed on rat sensory neurons in the indicated conditions for 8 h, showing the relative amount of TrkA peptides containing phosphorylated Y499 (Shc binding site) or Y683 and Y684 (activation loop tyrosines). (B) WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib (Foret) or 1 µM Lestaurtinib (Lest) for 10 h, probed for TrkA phosphorylated at Y683/Y684, and reprobed for total TrkA. (C) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of 1 µM lestaurtinib for 24 h and immunostained for βIII-tubulin (red) and counterstained with Hoechst 33258 (blue; bottom) and bright-field (top). The arrow indicates neuritic beading and the arrowhead an apoptotic cell. (D and E) Images as in C quantified for the number of swellings or beads (D) and the ratio of intact neurites (E). **, P
    Figure Legend Snippet: Foretinib and lestaurtinib inhibit the low levels of TrkA tyrosine phosphorylation in NGF-deprived neurons and suppress axon degeneration. (A) Quantitative normalized representation of the phosphoproteomics analysis performed on rat sensory neurons in the indicated conditions for 8 h, showing the relative amount of TrkA peptides containing phosphorylated Y499 (Shc binding site) or Y683 and Y684 (activation loop tyrosines). (B) WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib (Foret) or 1 µM Lestaurtinib (Lest) for 10 h, probed for TrkA phosphorylated at Y683/Y684, and reprobed for total TrkA. (C) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of 1 µM lestaurtinib for 24 h and immunostained for βIII-tubulin (red) and counterstained with Hoechst 33258 (blue; bottom) and bright-field (top). The arrow indicates neuritic beading and the arrowhead an apoptotic cell. (D and E) Images as in C quantified for the number of swellings or beads (D) and the ratio of intact neurites (E). **, P

    Techniques Used: Binding Assay, Activation Assay, Western Blot

    Foretinib inhibits local sympathetic axon degeneration caused by trophic factor deprivation. (A) Rat sympathetic neurons were switched into the indicated conditions for 48 h and immunostained for βIII-tubulin (white). Schematics on the left show the configurations used. In the top panels (i), NGF was maintained in the center compartment and removed from both side compartments, and 500 nM foretinib (Foret) added to one of the sides. In the center and bottom panels (ii and iii), NGF was withdrawn from all compartments, and foretinib added to one of the sides (ii) or one of the sides and the center compartment (iii). Fluorescence images show βIII-tubulin–positive axons in the two sides of the same compartments, and the bright-field images the cell bodies and proximal axons in the center compartments of the same cultures. Arrowhead denotes a shrunken, dying neuron. (B) Number of swellings or beads per 100 µm of neurite in the side compartments withdrawn from NGF with and without 500 nM foretinib. **, P
    Figure Legend Snippet: Foretinib inhibits local sympathetic axon degeneration caused by trophic factor deprivation. (A) Rat sympathetic neurons were switched into the indicated conditions for 48 h and immunostained for βIII-tubulin (white). Schematics on the left show the configurations used. In the top panels (i), NGF was maintained in the center compartment and removed from both side compartments, and 500 nM foretinib (Foret) added to one of the sides. In the center and bottom panels (ii and iii), NGF was withdrawn from all compartments, and foretinib added to one of the sides (ii) or one of the sides and the center compartment (iii). Fluorescence images show βIII-tubulin–positive axons in the two sides of the same compartments, and the bright-field images the cell bodies and proximal axons in the center compartments of the same cultures. Arrowhead denotes a shrunken, dying neuron. (B) Number of swellings or beads per 100 µm of neurite in the side compartments withdrawn from NGF with and without 500 nM foretinib. **, P

    Techniques Used: Fluorescence

    Foretinib suppresses the expression or activity of genes and proteins associated with sympathetic neuron death and degeneration and protects mitochondria. (A and B) Changes in gene expression in mouse sympathetic neurons withdrawn from NGF for 9 h ( bimEL , puma/bbc3 , trib3 , ddit3 , and hrk mRNAs) or 12 h ( pmaip1/noxa , gadd45γ , tap63 mRNAs) with or without foretinib (Foret) by quantitative RT-PCR; n ≥ 3 independent experiments. (C–G). WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib for 10–12 (C and F), 18 (D and E), or 24 (G) h. Blots were probed with antibodies to the indicated proteins, including phosphorylated (p) JNK, activated (a) Bax, cleaved caspase-3 (cc3), or αII-spectrin (cleaved [cl] and full-length [f.l.]), and reprobed for βIII-tubulin (tuj1), ERK1/2, or GAPDH to control for loading. (H and I) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of foretinib for 12 h, immunostained for cytochrome c (red, cyt-c) or βIII-tubulin (green), and quantified for neurons with diffuse cytochrome c (I), as indicated by arrows in H. (I) ***, P
    Figure Legend Snippet: Foretinib suppresses the expression or activity of genes and proteins associated with sympathetic neuron death and degeneration and protects mitochondria. (A and B) Changes in gene expression in mouse sympathetic neurons withdrawn from NGF for 9 h ( bimEL , puma/bbc3 , trib3 , ddit3 , and hrk mRNAs) or 12 h ( pmaip1/noxa , gadd45γ , tap63 mRNAs) with or without foretinib (Foret) by quantitative RT-PCR; n ≥ 3 independent experiments. (C–G). WB analysis of mouse sympathetic neurons withdrawn from NGF with or without 500 nM foretinib for 10–12 (C and F), 18 (D and E), or 24 (G) h. Blots were probed with antibodies to the indicated proteins, including phosphorylated (p) JNK, activated (a) Bax, cleaved caspase-3 (cc3), or αII-spectrin (cleaved [cl] and full-length [f.l.]), and reprobed for βIII-tubulin (tuj1), ERK1/2, or GAPDH to control for loading. (H and I) Mouse sympathetic neurons withdrawn from NGF in the presence or absence of foretinib for 12 h, immunostained for cytochrome c (red, cyt-c) or βIII-tubulin (green), and quantified for neurons with diffuse cytochrome c (I), as indicated by arrows in H. (I) ***, P

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot

    Foretinib prevents NGF-induced death and degeneration of sympathetic and sensory neurons. (A–D) Foretinib protects sympathetic neurons from degeneration more effectively than a JNK inhibitor. Murine sympathetic neurons were cultured in NGF for 2 d, withdrawn from NGF (−NGF), and cultured for 16 h with or without 2 µM JNK inhibitor TAT-TI-JIP 153–156 (JNKiVII), 500 nM foretinib (Foret), or NGF. Shown is immunostaining for βIII-tubulin (red) and costaining with Hoechst 33258 to highlight nuclear morphology (bottom) or bright-field (top) panels. Images were quantified as described in Materials and methods for the number of intact neurites (B), axonal beads (C), or condensed apoptotic nuclei (D). *, P
    Figure Legend Snippet: Foretinib prevents NGF-induced death and degeneration of sympathetic and sensory neurons. (A–D) Foretinib protects sympathetic neurons from degeneration more effectively than a JNK inhibitor. Murine sympathetic neurons were cultured in NGF for 2 d, withdrawn from NGF (−NGF), and cultured for 16 h with or without 2 µM JNK inhibitor TAT-TI-JIP 153–156 (JNKiVII), 500 nM foretinib (Foret), or NGF. Shown is immunostaining for βIII-tubulin (red) and costaining with Hoechst 33258 to highlight nuclear morphology (bottom) or bright-field (top) panels. Images were quantified as described in Materials and methods for the number of intact neurites (B), axonal beads (C), or condensed apoptotic nuclei (D). *, P

    Techniques Used: Cell Culture, Immunostaining

    TrkA inhibition partially rescues sympathetic neuron degeneration. (A) WB analysis of TrkA F592A sympathetic neurons withdrawn from NGF in the presence of 1 µM 1NMPP1 for 10 h, probed for Trk phosphorylated at Y683/Y684, and reprobed for total TrkA. (B) The specific TrkA F592A inhibitor 1NMPP1 partially rescues NGF withdrawal-induced degeneration. TrkA F592A sympathetic neurons cultured withdrawn from NGF in the presence or absence of 1 µM 1NMPP1 for 14 h and immunostained for βIII-tubulin and counterstained with Hoechst 33258. (C and D) Images as in B were quantified for the number of swellings or beads or for the ratio of intact neurites. (C) *, P
    Figure Legend Snippet: TrkA inhibition partially rescues sympathetic neuron degeneration. (A) WB analysis of TrkA F592A sympathetic neurons withdrawn from NGF in the presence of 1 µM 1NMPP1 for 10 h, probed for Trk phosphorylated at Y683/Y684, and reprobed for total TrkA. (B) The specific TrkA F592A inhibitor 1NMPP1 partially rescues NGF withdrawal-induced degeneration. TrkA F592A sympathetic neurons cultured withdrawn from NGF in the presence or absence of 1 µM 1NMPP1 for 14 h and immunostained for βIII-tubulin and counterstained with Hoechst 33258. (C and D) Images as in B were quantified for the number of swellings or beads or for the ratio of intact neurites. (C) *, P

    Techniques Used: Inhibition, Western Blot, Cell Culture

    23) Product Images from "NGF signaling in PC12 cells: the cooperation of p75NTR with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade"

    Article Title: NGF signaling in PC12 cells: the cooperation of p75NTR with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade

    Journal: Biology Open

    doi: 10.1242/bio.20135116

    Expression of p75 NTR and time-course of TrkA autophosphorylation at the Y751 and Y490 sites in PC12-27 cells transfected with the vector, empty (PC12-27/Ctrl) or including the full length p75 NTR (PC12-27/p75 NTR ). ( A ) The western blot of p75 NTR in wtPC12, PC12-27/Ctrl and PC12-27/p75 NTR cells. Quantization of the data, documenting the similar levels of the receptor in the wtPC12 and PC12-27/p75 NTR cells, is on the right. ( B ) The surface immunolocalization of p75 NTR in the PC12-27/Ctrl and PC12-27p75 NTR cells. The quantization of the results is on the right. Scale bar: 10 µm. ( C , D ) The time-course of the TrkA autophosphorylation at the Y751 and Y490 sites induced by NGF (100 ng/ml) in the two transfected subclones, PC12-27/Ctrl and PC12-27/p75 NTR . The quantization of these data is shown on the right. Statistical analysis and significance of the differences is given as specified in the legend for Fig. 1 .
    Figure Legend Snippet: Expression of p75 NTR and time-course of TrkA autophosphorylation at the Y751 and Y490 sites in PC12-27 cells transfected with the vector, empty (PC12-27/Ctrl) or including the full length p75 NTR (PC12-27/p75 NTR ). ( A ) The western blot of p75 NTR in wtPC12, PC12-27/Ctrl and PC12-27/p75 NTR cells. Quantization of the data, documenting the similar levels of the receptor in the wtPC12 and PC12-27/p75 NTR cells, is on the right. ( B ) The surface immunolocalization of p75 NTR in the PC12-27/Ctrl and PC12-27p75 NTR cells. The quantization of the results is on the right. Scale bar: 10 µm. ( C , D ) The time-course of the TrkA autophosphorylation at the Y751 and Y490 sites induced by NGF (100 ng/ml) in the two transfected subclones, PC12-27/Ctrl and PC12-27/p75 NTR . The quantization of these data is shown on the right. Statistical analysis and significance of the differences is given as specified in the legend for Fig. 1 .

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot

    Expression of TrkA and p75 NTR , and NGF-induced TrkA autophosphorylation responses in wtPC12 and PC12-27 cells. ( A , B ) The mRNA and protein of the two receptors revealed in the two clones by RT-PCR (A) and western blotting (B). Notice the lack of p75 NTR in the PC12-27 clone. ( C ) The surface immunolabeling of the two receptors in the wtPC12 and PC12-27 cells. The fractions of the total receptors distributed to the surface, given as percentages, are shown in the two subpanels below. Scale bars: 5 µm (left), 10 µm (right). ( D ) The time-course of the autophosphorylation induced by NGF (100 ng/ml) at three sites of the TrkA receptor, Y751 on the top, Y490 in the middle and Y670, Y674 and Y675, analyzed together, at the bottom. The time-course data of panel D are shown in quantitative terms in panel E . Here, and in the following figures, the number of gels analyzed quantitatively is given by the numbers written over the panels; the numbers flanking the gels are the MDa of the immunolabeled proteins, given only in the figure showing the protein for the first time. The significance of the results, given as averages ± s.e., is calculated with respect to the sample labeled 0 in each cell population. * P
    Figure Legend Snippet: Expression of TrkA and p75 NTR , and NGF-induced TrkA autophosphorylation responses in wtPC12 and PC12-27 cells. ( A , B ) The mRNA and protein of the two receptors revealed in the two clones by RT-PCR (A) and western blotting (B). Notice the lack of p75 NTR in the PC12-27 clone. ( C ) The surface immunolabeling of the two receptors in the wtPC12 and PC12-27 cells. The fractions of the total receptors distributed to the surface, given as percentages, are shown in the two subpanels below. Scale bars: 5 µm (left), 10 µm (right). ( D ) The time-course of the autophosphorylation induced by NGF (100 ng/ml) at three sites of the TrkA receptor, Y751 on the top, Y490 in the middle and Y670, Y674 and Y675, analyzed together, at the bottom. The time-course data of panel D are shown in quantitative terms in panel E . Here, and in the following figures, the number of gels analyzed quantitatively is given by the numbers written over the panels; the numbers flanking the gels are the MDa of the immunolabeled proteins, given only in the figure showing the protein for the first time. The significance of the results, given as averages ± s.e., is calculated with respect to the sample labeled 0 in each cell population. * P

    Techniques Used: Expressing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunolabeling, Multiple Displacement Amplification, Labeling

    ERK and PI3K signaling cascades in wtPC12 and PC12-27 cells. ( A ) The time-course of the ERK 1/2 phosphorylation induced by NGF (100 ng/ml) at the T202/Y204 sites analyzed together. ( B ) The phosphorylation responses induced at the T202/Y204 sites of ERK 1/2 in wtPC12 and PC12-27 cells kept for 1hr at rest (0), with NGF (N, 100 ng/ml), rapamycin (R, 1 µM) and the two together (NR). ( C ) The responses induced by the same treatments at the P-Akt(T308). In each cell sample the levels of the ERK (B), Akt and p75 NTR (C) proteins did not change during the experiments. The data on P-ERK 1/2(T202/Y204) and P-Akt(T308) are also shown in quantized terms on the right in panels B and C. The numbers flanking the gels are the MDa of the immunolabeled proteins. The statistical analysis and the significance of the differences are shown by the asterisks as specified in the legend for Fig. 1 .
    Figure Legend Snippet: ERK and PI3K signaling cascades in wtPC12 and PC12-27 cells. ( A ) The time-course of the ERK 1/2 phosphorylation induced by NGF (100 ng/ml) at the T202/Y204 sites analyzed together. ( B ) The phosphorylation responses induced at the T202/Y204 sites of ERK 1/2 in wtPC12 and PC12-27 cells kept for 1hr at rest (0), with NGF (N, 100 ng/ml), rapamycin (R, 1 µM) and the two together (NR). ( C ) The responses induced by the same treatments at the P-Akt(T308). In each cell sample the levels of the ERK (B), Akt and p75 NTR (C) proteins did not change during the experiments. The data on P-ERK 1/2(T202/Y204) and P-Akt(T308) are also shown in quantized terms on the right in panels B and C. The numbers flanking the gels are the MDa of the immunolabeled proteins. The statistical analysis and the significance of the differences are shown by the asterisks as specified in the legend for Fig. 1 .

    Techniques Used: Multiple Displacement Amplification, Immunolabeling

    Model of the changes of the NGF signaling induced in PC12-27 cells by the transfection of p75 NTR . The panel to the left illustrates the signaling in the non-transfected PC12-27 cells. Activation of TrkA by NGF binding induces a weak autophosphorylation at the Y490 site which is followed by a dissociation of the two signaling cascades, indicated by both the thickness of the arrows and the tone of the kinases: the ERK1/2 cascade is strong whereas the PI3K cascade is weak. The panel to the right shows that activation by NGF of the TrkA/p75 NTR complex induced a stronger autophosphorylation of Y490 accompanied by the parallel strong activation of both the cascades. In the first case the neurite outgrowth response remains low; in the second it is strong.
    Figure Legend Snippet: Model of the changes of the NGF signaling induced in PC12-27 cells by the transfection of p75 NTR . The panel to the left illustrates the signaling in the non-transfected PC12-27 cells. Activation of TrkA by NGF binding induces a weak autophosphorylation at the Y490 site which is followed by a dissociation of the two signaling cascades, indicated by both the thickness of the arrows and the tone of the kinases: the ERK1/2 cascade is strong whereas the PI3K cascade is weak. The panel to the right shows that activation by NGF of the TrkA/p75 NTR complex induced a stronger autophosphorylation of Y490 accompanied by the parallel strong activation of both the cascades. In the first case the neurite outgrowth response remains low; in the second it is strong.

    Techniques Used: Transfection, Activation Assay, Binding Assay

    Phenotype of the PC12-27/Ctrl and PC12-27/p75 NTR cells. ( A–D ) No difference exists between PC12-27/Ctrl and PC12-27/p75 NTR in a number of important features: the levels of the REST, TSC2 and β-catenin proteins (A); the β-catenin-dependent transcription revealed by a luciferase assay (B); the expression of the β-catenin-target gene, cMyc (C); the rate of cell proliferation (D). ( E ) Phase contrast images of the PC12-27/Ctrl and PC12-27/p75 NTR before and after a 48 hr treatment with NGF (100 ng/ml). Scale bar: 20 µm. ( F ) The expression of two neuronal markers, Map2 and β-III tubulin, in PC12-27/Ctrl and PC12-27/p75 NTR cells incubated for 48 hr with no treatment, with NGF (N, 100 ng/ml), rapamycin (R, 0.1 µM during the last 24 hr) and the two together.
    Figure Legend Snippet: Phenotype of the PC12-27/Ctrl and PC12-27/p75 NTR cells. ( A–D ) No difference exists between PC12-27/Ctrl and PC12-27/p75 NTR in a number of important features: the levels of the REST, TSC2 and β-catenin proteins (A); the β-catenin-dependent transcription revealed by a luciferase assay (B); the expression of the β-catenin-target gene, cMyc (C); the rate of cell proliferation (D). ( E ) Phase contrast images of the PC12-27/Ctrl and PC12-27/p75 NTR before and after a 48 hr treatment with NGF (100 ng/ml). Scale bar: 20 µm. ( F ) The expression of two neuronal markers, Map2 and β-III tubulin, in PC12-27/Ctrl and PC12-27/p75 NTR cells incubated for 48 hr with no treatment, with NGF (N, 100 ng/ml), rapamycin (R, 0.1 µM during the last 24 hr) and the two together.

    Techniques Used: Luciferase, Expressing, Incubation

    The ERK and PI3K cascades in the PC12-27/Ctrl and PC12-27/p75 NTR cells. ( A ) The time-course of ERK phosphorylation, slow in the PC12-27/Ctrl cells, faster in the PC12-27/p75 NTR cells. ( B , C ) The effects of 1 hr treatment with NGF (N, 100 ng/ml), rapamycin (R, 1 µM) and the two together (NR) on the P-ERK 1/2(T202/Y204) and P-Akt(T308) of the PC12-27/Ctrl and PC12-27/p75 NTR cells. The level of p75 NTR in the PC12-27/Ctrl and PC12-27/p75 NTR cells did not change in response to the treatments. The quantization of the P-ERK 1/2 (T202/Y204) and Akt(T308) phosphorylation data is shown in the panels below the gels. ( D ) The effects of the TrkA receptor inhibitor, Calbiochem 648450 (I, 10 nM, 2 hr), on the P-Akt(T308) and P-S6(S235/236) responses induced by NGF (N, 100 ng/ml), rapamycin (R, 1 µM) administered during the second hr, and the two together (NR). The inhibitor was found to have no appreciable effect on the small increase induced by NGF on the mTORC1 read-out which in contrast was blocked by rapamycin, as expected. In contrast, the inhibitor blocked the effect of NGF (but not that of rapamycin) on P-Akt(308), demonstrating the effect of the neurotrophin on the PI3K cascade to require the cooperation of both p75 NTR and TrkA. Statistical analysis of the differences is given as specified in the legend for Fig. 1 .
    Figure Legend Snippet: The ERK and PI3K cascades in the PC12-27/Ctrl and PC12-27/p75 NTR cells. ( A ) The time-course of ERK phosphorylation, slow in the PC12-27/Ctrl cells, faster in the PC12-27/p75 NTR cells. ( B , C ) The effects of 1 hr treatment with NGF (N, 100 ng/ml), rapamycin (R, 1 µM) and the two together (NR) on the P-ERK 1/2(T202/Y204) and P-Akt(T308) of the PC12-27/Ctrl and PC12-27/p75 NTR cells. The level of p75 NTR in the PC12-27/Ctrl and PC12-27/p75 NTR cells did not change in response to the treatments. The quantization of the P-ERK 1/2 (T202/Y204) and Akt(T308) phosphorylation data is shown in the panels below the gels. ( D ) The effects of the TrkA receptor inhibitor, Calbiochem 648450 (I, 10 nM, 2 hr), on the P-Akt(T308) and P-S6(S235/236) responses induced by NGF (N, 100 ng/ml), rapamycin (R, 1 µM) administered during the second hr, and the two together (NR). The inhibitor was found to have no appreciable effect on the small increase induced by NGF on the mTORC1 read-out which in contrast was blocked by rapamycin, as expected. In contrast, the inhibitor blocked the effect of NGF (but not that of rapamycin) on P-Akt(308), demonstrating the effect of the neurotrophin on the PI3K cascade to require the cooperation of both p75 NTR and TrkA. Statistical analysis of the differences is given as specified in the legend for Fig. 1 .

    Techniques Used:

    mTORC1 and mTORC2 in the PC12-27/Ctrl and PC12-27/p75 NTR cells; effects of the TrkA inhibitor. ( A ) The expression of p75 NTR does not change the responses of the mTORC1 read-out, P-S6(S325-326) to 1 hr treatment with NGF (N, 100 ng/ml). Rapamycin (R, 1 µM) induces inhibition of the mTORC1 read-out phosphorylation, which is more extensive in the PC12-27/Ctrl cells. ( B ) The p75 NTR transfection induces the rescue of the mTORC2 read-out P-Akt(S473) phosphorylation which is increased markedly by both NGF (N, 100 ng/ml) and rapamycin (R, 1 µM) The phosphorylation of another mTORC2 read-out, PKCα, was high in the PC12-27/p75 NTR cells already at rest, with no appreciable changes induced by the treatments. The quantized data of panels A and B in PC12-27/Ctrl and PC12-27/p75 NTR cells are given on the right panels. ( C ) Two hr treatment with the TrkA receptor inhibitor, Calbiochem 648450 (I, 10 nM), removed the response triggered in the PC12-27/p75 NTR cells by 1 hr treatment with NGF (N, 100 ng/ml), leaving however unchanged that triggered by rapamycin (R, 1 µM), administered alone or combined to NGF. These results demonstrate 1) that the NGF response is mediated by the cooperation of the TrkA and p75 NTR receptors; and 2) that the mTORC1.induced feed-back block by rapamycin cooperates with p75 NTR working however not at the TrkA receptor but at a post-receptor site. Statistical analysis of the differences on the right in panels A and B is given as specified in the legend for Fig. 1 .
    Figure Legend Snippet: mTORC1 and mTORC2 in the PC12-27/Ctrl and PC12-27/p75 NTR cells; effects of the TrkA inhibitor. ( A ) The expression of p75 NTR does not change the responses of the mTORC1 read-out, P-S6(S325-326) to 1 hr treatment with NGF (N, 100 ng/ml). Rapamycin (R, 1 µM) induces inhibition of the mTORC1 read-out phosphorylation, which is more extensive in the PC12-27/Ctrl cells. ( B ) The p75 NTR transfection induces the rescue of the mTORC2 read-out P-Akt(S473) phosphorylation which is increased markedly by both NGF (N, 100 ng/ml) and rapamycin (R, 1 µM) The phosphorylation of another mTORC2 read-out, PKCα, was high in the PC12-27/p75 NTR cells already at rest, with no appreciable changes induced by the treatments. The quantized data of panels A and B in PC12-27/Ctrl and PC12-27/p75 NTR cells are given on the right panels. ( C ) Two hr treatment with the TrkA receptor inhibitor, Calbiochem 648450 (I, 10 nM), removed the response triggered in the PC12-27/p75 NTR cells by 1 hr treatment with NGF (N, 100 ng/ml), leaving however unchanged that triggered by rapamycin (R, 1 µM), administered alone or combined to NGF. These results demonstrate 1) that the NGF response is mediated by the cooperation of the TrkA and p75 NTR receptors; and 2) that the mTORC1.induced feed-back block by rapamycin cooperates with p75 NTR working however not at the TrkA receptor but at a post-receptor site. Statistical analysis of the differences on the right in panels A and B is given as specified in the legend for Fig. 1 .

    Techniques Used: Expressing, Inhibition, Transfection, Blocking Assay

    Read-outs of mTORC1 (P-S6(S235/236)) and mTORC2 (P-Akt(S473)) in wtPC12 and PC12-27 cells. ( A , B ) wtPC12 and PC12-27 cells were treated for 48 hr with no stimulant (0), with NGF (N, 100 ng/ml), with rapamycin in the last 24 hr (R, 0.1 µM) and with the two together (NR). The quantization of the data is on the right panels. The levels of the S6 and Akt proteins were not changed by the treatments. The numbers flanking the gels are the MDa of the immunolabeled proteins. Statistical analysis and significance of the differences is given as specified in the legend for Fig. 1 .
    Figure Legend Snippet: Read-outs of mTORC1 (P-S6(S235/236)) and mTORC2 (P-Akt(S473)) in wtPC12 and PC12-27 cells. ( A , B ) wtPC12 and PC12-27 cells were treated for 48 hr with no stimulant (0), with NGF (N, 100 ng/ml), with rapamycin in the last 24 hr (R, 0.1 µM) and with the two together (NR). The quantization of the data is on the right panels. The levels of the S6 and Akt proteins were not changed by the treatments. The numbers flanking the gels are the MDa of the immunolabeled proteins. Statistical analysis and significance of the differences is given as specified in the legend for Fig. 1 .

    Techniques Used: Multiple Displacement Amplification, Immunolabeling

    24) Product Images from "Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation *"

    Article Title: Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.421669

    Ser 779 in the cytoplasmic tail of FGFR1 and FGFR2 is required for maximal ERK activation. A–D , PC12 cells expressing PFR1 or PFR1-S779G ( A ) or wt-FGFR2 or FGFR2-S779G ( B–D ) were stimulated with PDGF-BB ( A ), FGF9 ( B ), EGF ( C ), or NGF (
    Figure Legend Snippet: Ser 779 in the cytoplasmic tail of FGFR1 and FGFR2 is required for maximal ERK activation. A–D , PC12 cells expressing PFR1 or PFR1-S779G ( A ) or wt-FGFR2 or FGFR2-S779G ( B–D ) were stimulated with PDGF-BB ( A ), FGF9 ( B ), EGF ( C ), or NGF (

    Techniques Used: Activation Assay, Expressing

    25) Product Images from "Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection"

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004571

    SgG2 interacts with NGF. (A) Sensorgram showing the interaction of SgG2 with increasing concentrations of NGF. The concentrations of NGF are indicated at the right side of the sensorgram. (B) Sensorgram showing the interaction of SgG2 with NGF and the lack of interaction with TNF-α, IFN-α and IL-1. All analytes were injected at a 100 nM concentration. (C) Sensorgram depicting the interaction between increasing concentrations of SgG2 and NGF coupled on a sensor chip. As a negative control we used the same concentrations of purified HSV-2 gD. (D) Saturation curve for the binding of NGF to SgG2. The derived KD is shown (E) Sensorgram showing the interaction of coupled SgG2 with CXCL12β and NGF injected alone or in combination. In all cases the arrow indicates the end of injection. Abbreviations: Diff. Resp., Differential response; M, molar; R.U., response units; s, seconds.
    Figure Legend Snippet: SgG2 interacts with NGF. (A) Sensorgram showing the interaction of SgG2 with increasing concentrations of NGF. The concentrations of NGF are indicated at the right side of the sensorgram. (B) Sensorgram showing the interaction of SgG2 with NGF and the lack of interaction with TNF-α, IFN-α and IL-1. All analytes were injected at a 100 nM concentration. (C) Sensorgram depicting the interaction between increasing concentrations of SgG2 and NGF coupled on a sensor chip. As a negative control we used the same concentrations of purified HSV-2 gD. (D) Saturation curve for the binding of NGF to SgG2. The derived KD is shown (E) Sensorgram showing the interaction of coupled SgG2 with CXCL12β and NGF injected alone or in combination. In all cases the arrow indicates the end of injection. Abbreviations: Diff. Resp., Differential response; M, molar; R.U., response units; s, seconds.

    Techniques Used: Injection, Concentration Assay, Chromatin Immunoprecipitation, Negative Control, Purification, Binding Assay, Derivative Assay

    SgG2 reduces NGF induced TrkA internalization and retrograde transport. SCG dissociated neurons were grown during 5 DIV and deprived of NGF for 16 h. (A) Neurons were stimulated with NGF alone or together with the indicated viral proteins during 0, 15 and 120 min. TrkA internalization was analyzed by immunofluorescence without permeabilization and normalized for F-actin using phalloidin staining. Solid arrowhead points to a non-internalized cluster of TrkA at the plasma membrane. Images show a projection of at least 3 image planes. All images correspond to the same experiment, representative of two independent experiments ( n = 12 for each condition). Scale bar represents 5 μm. The graph shows the quantification of the TrkA/phalloidin staining at the plasma membrane over time in samples treated with NGF plus the indicated protein or buffer. (B) Mouse SCG dissociated neurons were grown during 7 DIV using microfluidic devices. Neurons were deprived of NGF for 16 h and NGF and viral proteins were added to the distal axon compartment during 120 min. TrkA phosphorylation was analyzed by immunofluorescence after permeabilization with Triton X-100 and normalized using phalloidin. The images and the graph correspond to the same experiment and are representative of three independent experiments. Scale bar 10 μm. n = 10 fields for each condition. A two-tailed unpaired T-test was used to calculate significance in (A) and (B) . *** P
    Figure Legend Snippet: SgG2 reduces NGF induced TrkA internalization and retrograde transport. SCG dissociated neurons were grown during 5 DIV and deprived of NGF for 16 h. (A) Neurons were stimulated with NGF alone or together with the indicated viral proteins during 0, 15 and 120 min. TrkA internalization was analyzed by immunofluorescence without permeabilization and normalized for F-actin using phalloidin staining. Solid arrowhead points to a non-internalized cluster of TrkA at the plasma membrane. Images show a projection of at least 3 image planes. All images correspond to the same experiment, representative of two independent experiments ( n = 12 for each condition). Scale bar represents 5 μm. The graph shows the quantification of the TrkA/phalloidin staining at the plasma membrane over time in samples treated with NGF plus the indicated protein or buffer. (B) Mouse SCG dissociated neurons were grown during 7 DIV using microfluidic devices. Neurons were deprived of NGF for 16 h and NGF and viral proteins were added to the distal axon compartment during 120 min. TrkA phosphorylation was analyzed by immunofluorescence after permeabilization with Triton X-100 and normalized using phalloidin. The images and the graph correspond to the same experiment and are representative of three independent experiments. Scale bar 10 μm. n = 10 fields for each condition. A two-tailed unpaired T-test was used to calculate significance in (A) and (B) . *** P

    Techniques Used: Immunofluorescence, Staining, Two Tailed Test

    SgG2 modifies NGF-TrkA signaling. Mouse SCG dissociated neurons were grown during 5 days in vitro (DIV). (A) Neurons were deprived of NGF for 16 h and were stimulated with NGF alone, NGF plus SgG2 or SgG2 alone during 5 min. TrkA-SgG2 interaction was analyzed by TrkA immunoprecipitation (IP) followed by Western blot (WB) to detect SgG2. Molecular sizes in kDa and the position of SgG2 (empty arrowhead) and TrkA (solid arrowhead) are indicated. The experiment shown is representative of three independent assays. (B-F) Neurons were deprived of NGF for 16 h and were stimulated with NGF and the indicated viral proteins during 0, 15 and 120 min. (B) The phosphorylation levels of TrkA, ERK, AKT and cofilin were analyzed by Western blot (WB) using specific antibodies. Detection of actin was used as a loading control. All blots correspond to the same experiment. Graphs show statistical analysis for (C) p-TrkA, (D) p-ERK, (E) pAKT and (F) p-cofilin levels. p-TrkA n = 4; p-ERK n = 4; p-AKT n = 4; p-cofilin n = 3. To calculate significance a two-tailed unpaired T-test was employed; * P
    Figure Legend Snippet: SgG2 modifies NGF-TrkA signaling. Mouse SCG dissociated neurons were grown during 5 days in vitro (DIV). (A) Neurons were deprived of NGF for 16 h and were stimulated with NGF alone, NGF plus SgG2 or SgG2 alone during 5 min. TrkA-SgG2 interaction was analyzed by TrkA immunoprecipitation (IP) followed by Western blot (WB) to detect SgG2. Molecular sizes in kDa and the position of SgG2 (empty arrowhead) and TrkA (solid arrowhead) are indicated. The experiment shown is representative of three independent assays. (B-F) Neurons were deprived of NGF for 16 h and were stimulated with NGF and the indicated viral proteins during 0, 15 and 120 min. (B) The phosphorylation levels of TrkA, ERK, AKT and cofilin were analyzed by Western blot (WB) using specific antibodies. Detection of actin was used as a loading control. All blots correspond to the same experiment. Graphs show statistical analysis for (C) p-TrkA, (D) p-ERK, (E) pAKT and (F) p-cofilin levels. p-TrkA n = 4; p-ERK n = 4; p-AKT n = 4; p-cofilin n = 3. To calculate significance a two-tailed unpaired T-test was employed; * P

    Techniques Used: In Vitro, Immunoprecipitation, Western Blot, Two Tailed Test

    SgG2 increases NGF-dependent axonal growth. (A,B,D) Mouse SCGs were grown as explants in collagen matrix in the presence of the indicated trophic factors and viral proteins. Neurons were stained with Tuj1 antibody targeting class III ß–tubulin. Nuclei were stained with TO-PRO-3. Images are a projection of at least 3 stacks, correspond to the same experiment, and are representative of three (A,D) or two (B) independent experiments. The graphs below represent the quantification from three (A,D) or two (B) independent experiments. To calculate significance, a two-tailed unpaired T-test was applied. *** P
    Figure Legend Snippet: SgG2 increases NGF-dependent axonal growth. (A,B,D) Mouse SCGs were grown as explants in collagen matrix in the presence of the indicated trophic factors and viral proteins. Neurons were stained with Tuj1 antibody targeting class III ß–tubulin. Nuclei were stained with TO-PRO-3. Images are a projection of at least 3 stacks, correspond to the same experiment, and are representative of three (A,D) or two (B) independent experiments. The graphs below represent the quantification from three (A,D) or two (B) independent experiments. To calculate significance, a two-tailed unpaired T-test was applied. *** P

    Techniques Used: Staining, Two Tailed Test

    SgG2 promotes the incorporation of TrkA into different microdomains of the plasma membrane. Mouse SCG dissociated neurons were grown during 5 DIV. Neurons were deprived of NGF for 16 h and subsequently stimulated with NGF, vCKBPs or both. Colocalization between TrkA and two different subtypes of lipid rafts was studied using immunofluorescence without permeabilization. TrkA colocalization with GM1 rafts 2 min (A) and 10 min (B) post-stimulation. TrkA colocalization with GM3 rafts 2 min (C) , and 10 min (D) post-stimulation. Confocal microscopy images correspond to one representative cell from each condition. The +ves image displays pseudocolored pixels from the areas within the plasma membrane in which both TrkA and the corresponding subtype of lipid rafts pixel value exceed the mean. Scale bar represents 5 μm. Graphs show the average values (mean±SEM) obtained for PC and ICQ. Plots in (A) represent n = 18–26 neurons from three independent assays. Plots in (B) show the results obtained for 15 cells from two independent experiments. Plots in (C) represent n = 21–35 neurons from three independent assays. Plots in (D) represent 16–23 neurons from two independent assays. Two-tailed unpaired T-test, * P
    Figure Legend Snippet: SgG2 promotes the incorporation of TrkA into different microdomains of the plasma membrane. Mouse SCG dissociated neurons were grown during 5 DIV. Neurons were deprived of NGF for 16 h and subsequently stimulated with NGF, vCKBPs or both. Colocalization between TrkA and two different subtypes of lipid rafts was studied using immunofluorescence without permeabilization. TrkA colocalization with GM1 rafts 2 min (A) and 10 min (B) post-stimulation. TrkA colocalization with GM3 rafts 2 min (C) , and 10 min (D) post-stimulation. Confocal microscopy images correspond to one representative cell from each condition. The +ves image displays pseudocolored pixels from the areas within the plasma membrane in which both TrkA and the corresponding subtype of lipid rafts pixel value exceed the mean. Scale bar represents 5 μm. Graphs show the average values (mean±SEM) obtained for PC and ICQ. Plots in (A) represent n = 18–26 neurons from three independent assays. Plots in (B) show the results obtained for 15 cells from two independent experiments. Plots in (C) represent n = 21–35 neurons from three independent assays. Plots in (D) represent 16–23 neurons from two independent assays. Two-tailed unpaired T-test, * P

    Techniques Used: Immunofluorescence, Confocal Microscopy, Two Tailed Test

    26) Product Images from "Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse"

    Article Title: Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22052589

    NGF attenuates CAPS tachyphylaxis. Response ratios (peak value obtained for second CAPS stimulation after [A]/peak value for first stimulation before [B] exposure to 100 ng/mL NGF) for the ( A ) number of responders or ( B – D ) Max. signal intensity evoked during the second exposure to 0.3 (blue) or 1 μM CAPS (red) divided by the values elicited the first time when the DRG were stimulated with the same concentration of agonist. In ( B – D ), the calculations were restricted to cells that conformed to the following criteria: ( B ) neurons that produced above threshold signals to 0.3 or 1 μM CAPS the first time each concentration was applied; note that both above and below threshold values elicited by the second stimulation with either agonist concentration were included in these analyses, ( C ) neurons excited above threshold both times the DRG were exposed to 0.3 or 1 μM CAPS, ( D ) all DRGNs excited at least one time by either the first or second stimulation with 0.3 or 1 μM CAPS; although the threshold criterion was used to identify the neurons to analyse, comparisons before and after NGF included values both above and below threshold. Paired two-tailed Student’s t-test were performed to compare the mean values of [A] and [B]; * p
    Figure Legend Snippet: NGF attenuates CAPS tachyphylaxis. Response ratios (peak value obtained for second CAPS stimulation after [A]/peak value for first stimulation before [B] exposure to 100 ng/mL NGF) for the ( A ) number of responders or ( B – D ) Max. signal intensity evoked during the second exposure to 0.3 (blue) or 1 μM CAPS (red) divided by the values elicited the first time when the DRG were stimulated with the same concentration of agonist. In ( B – D ), the calculations were restricted to cells that conformed to the following criteria: ( B ) neurons that produced above threshold signals to 0.3 or 1 μM CAPS the first time each concentration was applied; note that both above and below threshold values elicited by the second stimulation with either agonist concentration were included in these analyses, ( C ) neurons excited above threshold both times the DRG were exposed to 0.3 or 1 μM CAPS, ( D ) all DRGNs excited at least one time by either the first or second stimulation with 0.3 or 1 μM CAPS; although the threshold criterion was used to identify the neurons to analyse, comparisons before and after NGF included values both above and below threshold. Paired two-tailed Student’s t-test were performed to compare the mean values of [A] and [B]; * p

    Techniques Used: Concentration Assay, Produced, Two Tailed Test

    Brief exposure to NGF results in sensitisation to CAPS. Analysis tools were applied to signals evoked by 0.3 (blue dots) and 1 μM CAPS (red dots) before [B] and after [A] 5 min exposure to 100 ng/mL NGF (blue trace in Figure 1 A) to measure ( A ) Max. intensity, ( B ) signal duration and ( C ) lag time. Asterisks represent significance between the measurements before and after NGF treatment, determined by Student’s t-test for unpaired samples, unequal variance; * p
    Figure Legend Snippet: Brief exposure to NGF results in sensitisation to CAPS. Analysis tools were applied to signals evoked by 0.3 (blue dots) and 1 μM CAPS (red dots) before [B] and after [A] 5 min exposure to 100 ng/mL NGF (blue trace in Figure 1 A) to measure ( A ) Max. intensity, ( B ) signal duration and ( C ) lag time. Asterisks represent significance between the measurements before and after NGF treatment, determined by Student’s t-test for unpaired samples, unequal variance; * p

    Techniques Used:

    NGF evokes signals if applied before CAPS and excites a new cohort when re-applied afterwards; CAPS-induced signal strength correlates with responsiveness to NGF. ( A ) Summed increase in fluorescence from DRG treated, in the following order, with 100 ng/mL NGF (20 min followed by 40 min washout, [N1]); 1 μM CAPS (5 min, 15 min washout, [C1]); NGF for a second time (20 min, 20 min washout, [N2]); 1 μM CAPS again (5 min, 15 min washout, [C2]) and, finally, 10 μM CAPS (5 min, 35 min washout, [C3]). ( B ) Shows the relative increase in the number of active cells (number activated by 10 μM CAPS [C3]/number excited by 1 μM [C1]) calculated for all CAPS-excitable cells (Total) or only the subsets that were NGF-excitable and -refractory cells. ( C – E ) Neurons that responded during the first ([C1]; blue dots) or second exposure ([C2]; green dots) to 1 μM CAPS and those excited by 10 μM CAPS ([C3]; red dots) were sub-categorised according to whether they were excited both times (2, abscissa) on exposure to NGF (i.e., during [N1] and [N2]), only once (1, abscissa) by either application (i.e., [N1] or [N2]) or remained inactive both times (0, abscissa). Each sub-category was analysed for ( C ) lag time, ( D ) signal duration and ( E ) Max. intensity. Plotted data represents mean ± s.e.m. Asterisks indicate p values for Student’s t -tests compared between group 1 and the requisite data set in group 0, or between groups 1 and 2. ** p
    Figure Legend Snippet: NGF evokes signals if applied before CAPS and excites a new cohort when re-applied afterwards; CAPS-induced signal strength correlates with responsiveness to NGF. ( A ) Summed increase in fluorescence from DRG treated, in the following order, with 100 ng/mL NGF (20 min followed by 40 min washout, [N1]); 1 μM CAPS (5 min, 15 min washout, [C1]); NGF for a second time (20 min, 20 min washout, [N2]); 1 μM CAPS again (5 min, 15 min washout, [C2]) and, finally, 10 μM CAPS (5 min, 35 min washout, [C3]). ( B ) Shows the relative increase in the number of active cells (number activated by 10 μM CAPS [C3]/number excited by 1 μM [C1]) calculated for all CAPS-excitable cells (Total) or only the subsets that were NGF-excitable and -refractory cells. ( C – E ) Neurons that responded during the first ([C1]; blue dots) or second exposure ([C2]; green dots) to 1 μM CAPS and those excited by 10 μM CAPS ([C3]; red dots) were sub-categorised according to whether they were excited both times (2, abscissa) on exposure to NGF (i.e., during [N1] and [N2]), only once (1, abscissa) by either application (i.e., [N1] or [N2]) or remained inactive both times (0, abscissa). Each sub-category was analysed for ( C ) lag time, ( D ) signal duration and ( E ) Max. intensity. Plotted data represents mean ± s.e.m. Asterisks indicate p values for Student’s t -tests compared between group 1 and the requisite data set in group 0, or between groups 1 and 2. ** p

    Techniques Used: Fluorescence

    NGF evokes Ca2+ signals in a subset of CAPS-excitable cells. ( A ) Traces show increases in fluorescence intensity (summed from 3 experiments) in DRGNs exposed sequentially to CAPS for 5 min. (black bars, at concentrations indicated) and 100 ng/mL NGF for 5 (blue) or 20 min. (red). In the latter case, a 5 min. washout after the 20 min. exposure to NGF has been omitted from the figure to keep the subsequent responses to CAPS in register with those in the blue trace. Note that F 0 was re-zeroed (see Section 4 ) before each addition of CAPS or NGF. Examples of fluorescence traces from individual DRGNs are presented in Supplementary Figure S1 . ( B ) Shows the mean ± s.e.m. Max. fluorescence intensity increases in neurons that responded above threshold upon exposure to each stimulus, as indicated on abscissa, in DRG exposed to NGF for 5 (blue bars) or 20 min. (red bars). Significant differences were observed for 0.3 μM CAPS before NGF ( p = 0.03), reflecting minor variation between experimental groups, and for 1 μM CAPS after NGF ( p = 0.02) but this latter difference might be due to the high background fluorescence after 20 min. with NGF, which may interfere with the quantification of subsequent responses to CAPS. ( C ) Number of active cells counted in 1 min. intervals after the addition of NGF, plotted against time; data summed from 3 independent recordings for each NGF treatment. ( D ) Mean number of cells activated (± s.e.m., N = 3) over 20 min. after the addition of CAPS (at concentrations indicated), or NGF, in DRG exposed to the latter for 5 (blue) or 20 min. (red). Results obtained with either CAPS concentration are shown for a two-tailed Student’s t -test between groups exposed to NGF for 5 min. and those treated for 20 min; * p
    Figure Legend Snippet: NGF evokes Ca2+ signals in a subset of CAPS-excitable cells. ( A ) Traces show increases in fluorescence intensity (summed from 3 experiments) in DRGNs exposed sequentially to CAPS for 5 min. (black bars, at concentrations indicated) and 100 ng/mL NGF for 5 (blue) or 20 min. (red). In the latter case, a 5 min. washout after the 20 min. exposure to NGF has been omitted from the figure to keep the subsequent responses to CAPS in register with those in the blue trace. Note that F 0 was re-zeroed (see Section 4 ) before each addition of CAPS or NGF. Examples of fluorescence traces from individual DRGNs are presented in Supplementary Figure S1 . ( B ) Shows the mean ± s.e.m. Max. fluorescence intensity increases in neurons that responded above threshold upon exposure to each stimulus, as indicated on abscissa, in DRG exposed to NGF for 5 (blue bars) or 20 min. (red bars). Significant differences were observed for 0.3 μM CAPS before NGF ( p = 0.03), reflecting minor variation between experimental groups, and for 1 μM CAPS after NGF ( p = 0.02) but this latter difference might be due to the high background fluorescence after 20 min. with NGF, which may interfere with the quantification of subsequent responses to CAPS. ( C ) Number of active cells counted in 1 min. intervals after the addition of NGF, plotted against time; data summed from 3 independent recordings for each NGF treatment. ( D ) Mean number of cells activated (± s.e.m., N = 3) over 20 min. after the addition of CAPS (at concentrations indicated), or NGF, in DRG exposed to the latter for 5 (blue) or 20 min. (red). Results obtained with either CAPS concentration are shown for a two-tailed Student’s t -test between groups exposed to NGF for 5 min. and those treated for 20 min; * p

    Techniques Used: Fluorescence, Concentration Assay, Two Tailed Test

    Acute sensitisation by NGF of responses to CAPS is most prevalent in cells that exhibit [Ca2+]i signals when the neurotrophin is applied. Neurons that responded to 1 μM CAPS both times when it was applied before and after 5 min exposure to NGF (blue trace in Figure 1 A) were split into two groups depending on whether they additionally exhibited Ca 2+ signals in response to NGF alone (red dots, n = 13 neurons from 3 DRG recordings) or did not (blue dots, n = 170). The analysis tools were applied to measure ( A ) Max. intensity, ( B ) signal duration and ( C ) lag time for the CAPS-evoked signals before [B] (on abscissa) and after [A] the treatment with NGF. Asterisks show significant differences within groups between responses before and after NGF treatment (Student’s t -test, paired samples; * p
    Figure Legend Snippet: Acute sensitisation by NGF of responses to CAPS is most prevalent in cells that exhibit [Ca2+]i signals when the neurotrophin is applied. Neurons that responded to 1 μM CAPS both times when it was applied before and after 5 min exposure to NGF (blue trace in Figure 1 A) were split into two groups depending on whether they additionally exhibited Ca 2+ signals in response to NGF alone (red dots, n = 13 neurons from 3 DRG recordings) or did not (blue dots, n = 170). The analysis tools were applied to measure ( A ) Max. intensity, ( B ) signal duration and ( C ) lag time for the CAPS-evoked signals before [B] (on abscissa) and after [A] the treatment with NGF. Asterisks show significant differences within groups between responses before and after NGF treatment (Student’s t -test, paired samples; * p

    Techniques Used:

    NGF-excitable cells exhibit more robust responses to CAPS. Further analysis was performed on DRG subjected to the protocol described for the red trace in Figure 1 A. Cells were categorised according to whether or not they responded to 20 min. with NGF. ( A ) The number of NGF-excitable (red bars) and -refractory (blue bars) cells that responded to each treatment with the indicated CAPS concentrations applied before [B] or after [A] NGF. Analysis tools were applied to each sub-category to measure ( B ) Lag, ( C ) Duration and ( D ) Max. intensity, in all cases plotted as means ± s.e.m., with blue and red dots representing, respectively, values from individual cells assigned to the NGF-refractory and -excitable categories. Black asterisks represent p values derived by unpaired Student’s t-test between the NGF-excitable and –refractory cells within each CAPS stimulation group. Coloured asterisks show results from paired Student’s t-test for measurements of signals evoked by 1 μM CAPS before and after NGF, respectively, for each cell category; *** p
    Figure Legend Snippet: NGF-excitable cells exhibit more robust responses to CAPS. Further analysis was performed on DRG subjected to the protocol described for the red trace in Figure 1 A. Cells were categorised according to whether or not they responded to 20 min. with NGF. ( A ) The number of NGF-excitable (red bars) and -refractory (blue bars) cells that responded to each treatment with the indicated CAPS concentrations applied before [B] or after [A] NGF. Analysis tools were applied to each sub-category to measure ( B ) Lag, ( C ) Duration and ( D ) Max. intensity, in all cases plotted as means ± s.e.m., with blue and red dots representing, respectively, values from individual cells assigned to the NGF-refractory and -excitable categories. Black asterisks represent p values derived by unpaired Student’s t-test between the NGF-excitable and –refractory cells within each CAPS stimulation group. Coloured asterisks show results from paired Student’s t-test for measurements of signals evoked by 1 μM CAPS before and after NGF, respectively, for each cell category; *** p

    Techniques Used: Derivative Assay

    27) Product Images from "Long-Term Intranasal Nerve Growth Factor Treatment Favors Neuron Formation in de novo Brain Tissue"

    Article Title: Long-Term Intranasal Nerve Growth Factor Treatment Favors Neuron Formation in de novo Brain Tissue

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2022.871532

    Histology of brain sections and characterization of the reconstructed tissue and edge of the lesion: glio- and angiogensesis. Representative images and quantification of GFAP (A,D) , Iba1 (B,E) and PDGFRB (C,F) mean intensity in PS ( left ) and NGF ( middle ) groups. There was no significant difference between the groups, as reported in the graphs (D–F) . All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7 Scale bars: 50 μm. PS: physiological saline; NGF: nerve growth factor; GFAP: glial fibrillary acidic protein; PDGFRB: platelet-derived growth factor receptor beta; L: lesion; RT: reconstructed tissue; HT: healthy tissue.
    Figure Legend Snippet: Histology of brain sections and characterization of the reconstructed tissue and edge of the lesion: glio- and angiogensesis. Representative images and quantification of GFAP (A,D) , Iba1 (B,E) and PDGFRB (C,F) mean intensity in PS ( left ) and NGF ( middle ) groups. There was no significant difference between the groups, as reported in the graphs (D–F) . All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7 Scale bars: 50 μm. PS: physiological saline; NGF: nerve growth factor; GFAP: glial fibrillary acidic protein; PDGFRB: platelet-derived growth factor receptor beta; L: lesion; RT: reconstructed tissue; HT: healthy tissue.

    Techniques Used: Derivative Assay

    Pie chart showing the percent of cell types detected in the brain sections of PS ( left ) and NGF ( right ) groups identified by histology 12 weeks post-injury. PS group: n = 6; NGF group: n = 7. PS: physiological saline; NGF: nerve growth factor; DCX: doublecortin; NeuN: neuronal nuclei; GFAP: glial fibrillary acidic protein; PDGFRB: platelet-derived growth factor receptor beta; β3-Tub: beta 3 tubulin.
    Figure Legend Snippet: Pie chart showing the percent of cell types detected in the brain sections of PS ( left ) and NGF ( right ) groups identified by histology 12 weeks post-injury. PS group: n = 6; NGF group: n = 7. PS: physiological saline; NGF: nerve growth factor; DCX: doublecortin; NeuN: neuronal nuclei; GFAP: glial fibrillary acidic protein; PDGFRB: platelet-derived growth factor receptor beta; β3-Tub: beta 3 tubulin.

    Techniques Used: Derivative Assay

    Behavioral tests following injury to the sensorimotor cortex. (A) Grip strength test shows the grip strength of the front paw contralateral to the injected hemisphere compared to the other paw, expressed as %. The Wilcoxon test compares the values measured 2 days post- compared to the pre-injury ones for the PS (gray triangles) and NGF (light yellow squares) groups (** p
    Figure Legend Snippet: Behavioral tests following injury to the sensorimotor cortex. (A) Grip strength test shows the grip strength of the front paw contralateral to the injected hemisphere compared to the other paw, expressed as %. The Wilcoxon test compares the values measured 2 days post- compared to the pre-injury ones for the PS (gray triangles) and NGF (light yellow squares) groups (** p

    Techniques Used: Injection

    Detailed study protocol. Behavioral tests started before injury and were performed at time points: day 2 and 4, and week 2, 3, 4, 8, and 12 post-injury. Intranasal administration of PS (vehicle) or NGF started at day 10 post-injury and lasted for 10 weeks (5 weeks + 5 weeks with 2 weeks of wash-out). MRI acquisitions were performed at day 1- and 8-weeks or 12-weeks post-injury. The gray arrow indicates stroke evolution: acute phase until day 7, early subacute phase from day 7 to week 8 post-injury; mid subacute phase from week 8 to 12 post-injury. Each light blue-dash indicates one week. Yellow oblique-parallel lines on the gray arrow indicate the wash-out period. PS: physiological saline; NGF: nerve growth factor; MRI: magnetic resonance imaging; BrdU: 5′-bromo-2-deoxyuridine.
    Figure Legend Snippet: Detailed study protocol. Behavioral tests started before injury and were performed at time points: day 2 and 4, and week 2, 3, 4, 8, and 12 post-injury. Intranasal administration of PS (vehicle) or NGF started at day 10 post-injury and lasted for 10 weeks (5 weeks + 5 weeks with 2 weeks of wash-out). MRI acquisitions were performed at day 1- and 8-weeks or 12-weeks post-injury. The gray arrow indicates stroke evolution: acute phase until day 7, early subacute phase from day 7 to week 8 post-injury; mid subacute phase from week 8 to 12 post-injury. Each light blue-dash indicates one week. Yellow oblique-parallel lines on the gray arrow indicate the wash-out period. PS: physiological saline; NGF: nerve growth factor; MRI: magnetic resonance imaging; BrdU: 5′-bromo-2-deoxyuridine.

    Techniques Used: Magnetic Resonance Imaging

    MRI at 24 h (vertical section 1), 8 and 12 weeks (section 2) and Histology of brain sections (section 3) and characterization of the lesion: the reconstructed tissue at 8 and 12 weeks post-injury. 1. At 24 h post-lesion, T2 weighted and diffusion weighted images show hyperintense edematous regions, vasogenic and cytotoxic edema, respectively. Diffusion images were not acquired for all rats at 24 h. Lesions are characterized by an increase in T2 signal and an decrease in water diffusion (hyperintense area on diffusion-weighted images). 2. As tissue becomes necrotic, T2 and water diffusion increase. The reconstructed tissue was observed both on MRI slices and at the exact same location on histology sections (red arrowheads). Restricted water diffusion is evidenced by hyperintensities in the core of the reconstructed tissue (yellow arrowheads). 3. Nissl staining on two coronal sections separated by 240 μm and the corresponding MRI slice (section 2, thickness 300 μm ) showing the reconstructed tissue. (A) Brain images from two rats sacrificed at 8 weeks are shown: PS rat (upper panel) and NGF rat (lower panel). Axial images are shown for the PS rat because a MRI artifact, probably coming from a blood clot, caused a distortion in the coronal slices near the skull. Reconstructed tissue is detectable in PS rat, while it is not in NGF rat, because the ventricle is dilated and reaches the skull. In the perilesional area of NGF rat, a hypointense area on T2 images appears (green arrowhead), compatible with newly generated cells (DCX+ and BrdU+ cells, data not shown). (B) Brain images from four different rats that were sacrificed at 12 weeks post-injury are shown. The upper panel shows PS group, while the lower three panels the NGF group. For the PS rat, thin filaments of reconstructed tissue could be detected by MRI (red arrowheads). Diffusion weighted images corresponding to the same section are shown on the right. Histological assay may damage the fragile neotissue (Nissl staining). Scale bars: 5 mm. MRI: magnetic resonance imaging; PS: physiological saline; NGF: Nerve Growth Factor; DCX: doublecortin and BrdU: BrdU: 5′-bromo-2-deoxyuridine.
    Figure Legend Snippet: MRI at 24 h (vertical section 1), 8 and 12 weeks (section 2) and Histology of brain sections (section 3) and characterization of the lesion: the reconstructed tissue at 8 and 12 weeks post-injury. 1. At 24 h post-lesion, T2 weighted and diffusion weighted images show hyperintense edematous regions, vasogenic and cytotoxic edema, respectively. Diffusion images were not acquired for all rats at 24 h. Lesions are characterized by an increase in T2 signal and an decrease in water diffusion (hyperintense area on diffusion-weighted images). 2. As tissue becomes necrotic, T2 and water diffusion increase. The reconstructed tissue was observed both on MRI slices and at the exact same location on histology sections (red arrowheads). Restricted water diffusion is evidenced by hyperintensities in the core of the reconstructed tissue (yellow arrowheads). 3. Nissl staining on two coronal sections separated by 240 μm and the corresponding MRI slice (section 2, thickness 300 μm ) showing the reconstructed tissue. (A) Brain images from two rats sacrificed at 8 weeks are shown: PS rat (upper panel) and NGF rat (lower panel). Axial images are shown for the PS rat because a MRI artifact, probably coming from a blood clot, caused a distortion in the coronal slices near the skull. Reconstructed tissue is detectable in PS rat, while it is not in NGF rat, because the ventricle is dilated and reaches the skull. In the perilesional area of NGF rat, a hypointense area on T2 images appears (green arrowhead), compatible with newly generated cells (DCX+ and BrdU+ cells, data not shown). (B) Brain images from four different rats that were sacrificed at 12 weeks post-injury are shown. The upper panel shows PS group, while the lower three panels the NGF group. For the PS rat, thin filaments of reconstructed tissue could be detected by MRI (red arrowheads). Diffusion weighted images corresponding to the same section are shown on the right. Histological assay may damage the fragile neotissue (Nissl staining). Scale bars: 5 mm. MRI: magnetic resonance imaging; PS: physiological saline; NGF: Nerve Growth Factor; DCX: doublecortin and BrdU: BrdU: 5′-bromo-2-deoxyuridine.

    Techniques Used: Magnetic Resonance Imaging, Diffusion-based Assay, Staining, Generated

    Characterization of the lesion. (A) Coronal section corresponding to the malonate injection site (red asterisk) and lesion size (red dashed line), adapted from Paxinos Atlas. (B) Representative T2-weighted MRI color-coded images showing the localization of the lesion in the two groups of rats (PS: left panel ; NGF: right panel ). The panels show the overlap map of injured voxels, providing an overview of the lesioned brain areas 24 h after the injection of malonate for the groups. Color indicates the number of rats injured at each voxel. (C) Graph showing the correlation between the lesion volume 24 h after the injection of malonate and motor deficits (NSS score) measured 48 h post-injury ( p
    Figure Legend Snippet: Characterization of the lesion. (A) Coronal section corresponding to the malonate injection site (red asterisk) and lesion size (red dashed line), adapted from Paxinos Atlas. (B) Representative T2-weighted MRI color-coded images showing the localization of the lesion in the two groups of rats (PS: left panel ; NGF: right panel ). The panels show the overlap map of injured voxels, providing an overview of the lesioned brain areas 24 h after the injection of malonate for the groups. Color indicates the number of rats injured at each voxel. (C) Graph showing the correlation between the lesion volume 24 h after the injection of malonate and motor deficits (NSS score) measured 48 h post-injury ( p

    Techniques Used: Injection, Magnetic Resonance Imaging

    Histology of brain sections and characterization of the lesion: the glial scar and the reconstructed tissue at 12 weeks post-injury. (A) Quantification of the glial scar thickness (expressed in μm) based on GFAP mean intensity (fluorescence) in PS and NGF groups. There was no significant difference between the groups. (B) Quantification showing the microglia (Iba1 mean intensity) in the glial scar in PS and NGF groups. There was no significant difference between the groups. (C,D) Nissl staining on coronal sections ( left ) and magnifications ( middle ) showing the edges of the lesion (black dashed lines) and the reconstructed tissue (red dotted lines) 12 weeks post-injury in PS (C) and NGF (D) groups. A migration pathway is observed between the ventricle and the lesion edge [Panel (C,D) , Red arrow]. (E) magnifications showing pyknotic tissue (green arrowhead) and migration pathway (red arrow). (F): Graph showing the quantification of the reconstructed tissue (RT) normalized by the lesion volume in PS and NGF groups. All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7. Scale bars: 5 mm. PS: physiological saline; NGF: nerve growth factor; GFAP: glial fibrillary acidic protein; a.u: arbitrary unit.
    Figure Legend Snippet: Histology of brain sections and characterization of the lesion: the glial scar and the reconstructed tissue at 12 weeks post-injury. (A) Quantification of the glial scar thickness (expressed in μm) based on GFAP mean intensity (fluorescence) in PS and NGF groups. There was no significant difference between the groups. (B) Quantification showing the microglia (Iba1 mean intensity) in the glial scar in PS and NGF groups. There was no significant difference between the groups. (C,D) Nissl staining on coronal sections ( left ) and magnifications ( middle ) showing the edges of the lesion (black dashed lines) and the reconstructed tissue (red dotted lines) 12 weeks post-injury in PS (C) and NGF (D) groups. A migration pathway is observed between the ventricle and the lesion edge [Panel (C,D) , Red arrow]. (E) magnifications showing pyknotic tissue (green arrowhead) and migration pathway (red arrow). (F): Graph showing the quantification of the reconstructed tissue (RT) normalized by the lesion volume in PS and NGF groups. All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7. Scale bars: 5 mm. PS: physiological saline; NGF: nerve growth factor; GFAP: glial fibrillary acidic protein; a.u: arbitrary unit.

    Techniques Used: Fluorescence, Staining, Migration

    Flowchart of the study, with the details on the rats excluded from the protocol: n = 4 died after the surgery (excessive edema); n = 1 excluded because of light deficit and performance > 60% at the grip strength test. Rats were sacrificed at 8 weeks ( n = 2 per group) and 12 weeks ( n = 7 per group) to evaluate the presence of reconstructed tissue at the different time points. PS: physiological saline; NGF: nerve growth factor; MRI: Magnetic Resonance Imaging.
    Figure Legend Snippet: Flowchart of the study, with the details on the rats excluded from the protocol: n = 4 died after the surgery (excessive edema); n = 1 excluded because of light deficit and performance > 60% at the grip strength test. Rats were sacrificed at 8 weeks ( n = 2 per group) and 12 weeks ( n = 7 per group) to evaluate the presence of reconstructed tissue at the different time points. PS: physiological saline; NGF: nerve growth factor; MRI: Magnetic Resonance Imaging.

    Techniques Used: Magnetic Resonance Imaging

    Histology of brain sections and characterization of the reconstructed tissue neurogenesis. Representative images and quantification of DCX (A,F) , beta 3 tubulin (D,I) mean intensity, NeuN (E,J) cell number (percentage of DAPI+ cells) and BrdU (B,H) cell number (percentage of DAPI+ cells) in the reconstructed tissue in PS ( left ) and NGF ( middle ) groups. Double staining with BrdU/DCX and BrdU/NeuN is shown in panel (C,E) (inserts). (C) Representative image showing BrdU (green) and DCX (red) staining. The inserts show the cells expressing both markers (white arrowheads). (E) Representative image showing NeuN (red) staining. The inserts show nuclei positives for BrdU (green) and NeuN markers (white arrowheads). No significant difference was observed for DCX, beta 3 tubulin and BrdU between the groups, as reported in the graphs (F–I) . A significant difference in percentage of NeuN positive-cells was observed between the groups (## p = 0.0043). All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7 except for BrdU. Scale bars: 50 μm. PS: physiological saline; NGF: nerve growth factor; DCX: doublecortin; NeuN: neuronal nuclei; BrdU: 5-bromo-2-deoxyuridine. L: lesion; RT: reconstructed tissue; HT: healthy tissue.
    Figure Legend Snippet: Histology of brain sections and characterization of the reconstructed tissue neurogenesis. Representative images and quantification of DCX (A,F) , beta 3 tubulin (D,I) mean intensity, NeuN (E,J) cell number (percentage of DAPI+ cells) and BrdU (B,H) cell number (percentage of DAPI+ cells) in the reconstructed tissue in PS ( left ) and NGF ( middle ) groups. Double staining with BrdU/DCX and BrdU/NeuN is shown in panel (C,E) (inserts). (C) Representative image showing BrdU (green) and DCX (red) staining. The inserts show the cells expressing both markers (white arrowheads). (E) Representative image showing NeuN (red) staining. The inserts show nuclei positives for BrdU (green) and NeuN markers (white arrowheads). No significant difference was observed for DCX, beta 3 tubulin and BrdU between the groups, as reported in the graphs (F–I) . A significant difference in percentage of NeuN positive-cells was observed between the groups (## p = 0.0043). All the graphs show the distribution of individual values and the median and the interquartile range. PS group: n = 6; NGF group: n = 7 except for BrdU. Scale bars: 50 μm. PS: physiological saline; NGF: nerve growth factor; DCX: doublecortin; NeuN: neuronal nuclei; BrdU: 5-bromo-2-deoxyuridine. L: lesion; RT: reconstructed tissue; HT: healthy tissue.

    Techniques Used: Double Staining, Staining, Expressing

    28) Product Images from "Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons"

    Article Title: Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons

    Journal: Cell death and differentiation

    doi: 10.1038/cdd.2008.76

    IKKs are necessary for NGF- and GDNF-mediated survival of sympathetic neurons. (A and B) Neurons were infected with shRNAs to IKKs in combination (“α+ β”) or alone (“α” or “β”)
    Figure Legend Snippet: IKKs are necessary for NGF- and GDNF-mediated survival of sympathetic neurons. (A and B) Neurons were infected with shRNAs to IKKs in combination (“α+ β”) or alone (“α” or “β”)

    Techniques Used: Infection

    Neither PI 3-K/Akt nor ERK1/2 are necessary for GDNF-mediated survival. (A) Sympathetic neurons from wild type mice were cultured for five days in NGF and then switched to GDNF with or without the indicated doses of the PI 3-K inhibitor LY294002. Neuronal
    Figure Legend Snippet: Neither PI 3-K/Akt nor ERK1/2 are necessary for GDNF-mediated survival. (A) Sympathetic neurons from wild type mice were cultured for five days in NGF and then switched to GDNF with or without the indicated doses of the PI 3-K inhibitor LY294002. Neuronal

    Techniques Used: Mouse Assay, Cell Culture

    Knockdown of B-Raf, but not A-Raf or C-Raf, prevents GDNF-and NGF-mediated survival of wild type mouse sympathetic neurons. (A,B,C) Left panels, neurons were infected with lentiviruses expressing shRNAs to A-Raf (A1 and A2), B-Raf (B1 and B2) and C-Raf
    Figure Legend Snippet: Knockdown of B-Raf, but not A-Raf or C-Raf, prevents GDNF-and NGF-mediated survival of wild type mouse sympathetic neurons. (A,B,C) Left panels, neurons were infected with lentiviruses expressing shRNAs to A-Raf (A1 and A2), B-Raf (B1 and B2) and C-Raf

    Techniques Used: Infection, Expressing

    29) Product Images from "Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1"

    Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202101075

    Model. Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of EVs exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neurons. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mESCs (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.
    Figure Legend Snippet: Model. Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of EVs exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neurons. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mESCs (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.

    Techniques Used:

    EV production increased during neuronal differentiation. (A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.
    Figure Legend Snippet: EV production increased during neuronal differentiation. (A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.

    Techniques Used: Purification, Staining, Cell Culture, Western Blot

    RA-induced EVs promote mESC neural fate commitment. (A) The cellular morphology of PC12 cells untreated or treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 µm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells untreated and treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments (*, P
    Figure Legend Snippet: RA-induced EVs promote mESC neural fate commitment. (A) The cellular morphology of PC12 cells untreated or treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 µm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells untreated and treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments (*, P

    Techniques Used: Expressing

    EVs show different buoyant density distribution during PC12 neuronal differentiation. (A) The cellular morphology of PC12 cells cultured in growth medium or low-serum medium with NGF (50 ng/ml) for 3, 6, and 9 d. Scale bars, 50 µm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low-serum medium without (Control) or with different doses of NGF (50 and 100 ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted are from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P
    Figure Legend Snippet: EVs show different buoyant density distribution during PC12 neuronal differentiation. (A) The cellular morphology of PC12 cells cultured in growth medium or low-serum medium with NGF (50 ng/ml) for 3, 6, and 9 d. Scale bars, 50 µm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low-serum medium without (Control) or with different doses of NGF (50 and 100 ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted are from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P
    Figure Legend Snippet: Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P

    Techniques Used: Incubation, Purification

    30) Product Images from "B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS"

    Article Title: B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131780

    Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.
    Figure Legend Snippet: Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue β-gal–positive (presumptive TrkA + ) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA −/− embryo (bottom; compare with middle for TrkA −/− alone) is similar to that seen in a control TrkA WT/− littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA −/− background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA + afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA + trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.

    Techniques Used: Expressing, Mouse Assay

    Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.
    Figure Legend Snippet: Activation of B-RAF indirectly rescues CGRP expression in TrkA −/− nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling ( LSL-kaBraf:nes-Cre:TrkA −/− :Bax −/− ). Arrowhead indicates the CGRP + motor neurons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax −/− :nes-Cre DRG neurons. Images are representative of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per genotype. Bars: (A and B) 100 µm; (C) 20 µm.

    Techniques Used: Activation Assay, Expressing, Staining, Mouse Assay, Cell Culture, In Vitro

    31) Product Images from "Regulation of neuronal survival by the extracellular signal-regulated protein kinase 5"

    Article Title: Regulation of neuronal survival by the extracellular signal-regulated protein kinase 5

    Journal: Cell death and differentiation

    doi: 10.1038/cdd.2008.193

    ERK5 regulates the transcription of bad via RSK-dependent CREB phosphorylation. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. b - d , PC6.3 cells were transiently transfected with a wild type or with a CRE-deficient (mt) Bad-luciferase construct together with (+) or without (−) DN-ERK5. The following day the cells were cultured in differentiating medium containing NGF (+) for a further 36 hours. Where indicated, NGF was removed (−) 18 h prior to the cells being harvested. Extracts were analyzed for the expression and the phosphorylation (P) of RSK and of CREB by immunoblot ( a , b ). Chromatin was immunoprecipitated with an antibody to CREB or to an irrelevant protein (Ctrl) to monitor the non-specific binding to the beads. The precipitated DNA was amplified by semi-quantitative PCR ( c ) or by RT PCR ( d ). Input DNA levels were used to monitor transfection efficiency. The data correspond to the mean ± SE of three independent experiments and are normalized to input DNA levels.
    Figure Legend Snippet: ERK5 regulates the transcription of bad via RSK-dependent CREB phosphorylation. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. b - d , PC6.3 cells were transiently transfected with a wild type or with a CRE-deficient (mt) Bad-luciferase construct together with (+) or without (−) DN-ERK5. The following day the cells were cultured in differentiating medium containing NGF (+) for a further 36 hours. Where indicated, NGF was removed (−) 18 h prior to the cells being harvested. Extracts were analyzed for the expression and the phosphorylation (P) of RSK and of CREB by immunoblot ( a , b ). Chromatin was immunoprecipitated with an antibody to CREB or to an irrelevant protein (Ctrl) to monitor the non-specific binding to the beads. The precipitated DNA was amplified by semi-quantitative PCR ( c ) or by RT PCR ( d ). Input DNA levels were used to monitor transfection efficiency. The data correspond to the mean ± SE of three independent experiments and are normalized to input DNA levels.

    Techniques Used: Infection, Cell Culture, Transfection, Luciferase, Construct, Expressing, Immunoprecipitation, Binding Assay, Amplification, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Regulation of neuronal survival by the ERK5 cascade. The requirement of ERK5 to phosphorylate PKB at Ser473 prevents the nuclear translocation of the pro-apoptotic transcription factor Foxo3a and thereby inhibits Bim expression. ERK5 also mediates the phosphorylation of RSK in response to NGF. In the nucleus, RSK stimulates the transcriptional activity of CREB. CREB is a pro-survival transcription factor that can inhibit the transcription of genes responsible for apoptosis including Bad. The binding of CREB to the CRE sites in the bad -promoter is dependent on ERK5.
    Figure Legend Snippet: Regulation of neuronal survival by the ERK5 cascade. The requirement of ERK5 to phosphorylate PKB at Ser473 prevents the nuclear translocation of the pro-apoptotic transcription factor Foxo3a and thereby inhibits Bim expression. ERK5 also mediates the phosphorylation of RSK in response to NGF. In the nucleus, RSK stimulates the transcriptional activity of CREB. CREB is a pro-survival transcription factor that can inhibit the transcription of genes responsible for apoptosis including Bad. The binding of CREB to the CRE sites in the bad -promoter is dependent on ERK5.

    Techniques Used: Translocation Assay, Expressing, Activity Assay, Binding Assay

    ERK5, ERK1/2 and PKB are required to support the survival of NGF-dependent SCG neurons. Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured for a further 48 hours in presence of NGF (50 ng/ml). Where indicated, the cells were treated with UO126 (10 μM), wortmannin (50 nM), or PD0325901 (25 nM) 6 h after the infection. The drugs were replaced every 12 h for the remaining time of the infection. a , c , Extracts were analyzed for ERK5, ERK1/2 and PKB expression, and for phosphorylation (P) of ERK1/2 and of PKB at Thr308 by immunoblot. The electrophoretic mobility shift caused by the phosphorylation of ERK5 is indicated by an arrow. Similar results were obtained in two independent experiments. b , Caspase 3 activity was measured by caspase assay. The data correspond to the mean ± SE of three independent experiments performed in duplicate. d , Cell survival was measured by LDH assay. The data correspond to the mean ± range of two independent experiments performed in duplicate. *, P
    Figure Legend Snippet: ERK5, ERK1/2 and PKB are required to support the survival of NGF-dependent SCG neurons. Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured for a further 48 hours in presence of NGF (50 ng/ml). Where indicated, the cells were treated with UO126 (10 μM), wortmannin (50 nM), or PD0325901 (25 nM) 6 h after the infection. The drugs were replaced every 12 h for the remaining time of the infection. a , c , Extracts were analyzed for ERK5, ERK1/2 and PKB expression, and for phosphorylation (P) of ERK1/2 and of PKB at Thr308 by immunoblot. The electrophoretic mobility shift caused by the phosphorylation of ERK5 is indicated by an arrow. Similar results were obtained in two independent experiments. b , Caspase 3 activity was measured by caspase assay. The data correspond to the mean ± SE of three independent experiments performed in duplicate. d , Cell survival was measured by LDH assay. The data correspond to the mean ± range of two independent experiments performed in duplicate. *, P

    Techniques Used: Infection, Cell Culture, Expressing, Electrophoretic Mobility Shift Assay, Activity Assay, Caspase Assay, Lactate Dehydrogenase Assay

    ERK5 is required to suppress the expression of Bad and Bim. Homozygous flox SCG neurons were infected with a control adenovirus (GFP or LacZ) or with an adenovirus encoding Cre. In e and f , the cells were infected 1 h later with lentiviruses encoding control, bim or bad shRNA. The neurons were cultured for a further 48 hours in presence of NGF (50 ng/ml). a , c - e , Extracts were analyzed for Bad, Bim and Bid expression by immunoblot. The detection of tubulin expression was performed to monitor protein loading. Images of Bad and Bim are from the same samples. Immunoblot signals were quantified with the ImageQuantifier software (BioImage, Jackson MI). The data correspond to the mean ± SE of three independent experiments. *, P
    Figure Legend Snippet: ERK5 is required to suppress the expression of Bad and Bim. Homozygous flox SCG neurons were infected with a control adenovirus (GFP or LacZ) or with an adenovirus encoding Cre. In e and f , the cells were infected 1 h later with lentiviruses encoding control, bim or bad shRNA. The neurons were cultured for a further 48 hours in presence of NGF (50 ng/ml). a , c - e , Extracts were analyzed for Bad, Bim and Bid expression by immunoblot. The detection of tubulin expression was performed to monitor protein loading. Images of Bad and Bim are from the same samples. Immunoblot signals were quantified with the ImageQuantifier software (BioImage, Jackson MI). The data correspond to the mean ± SE of three independent experiments. *, P

    Techniques Used: Expressing, Infection, shRNA, Cell Culture, Software

    ERK5 regulates Bad and Bim transcription. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. Total RNA was extracted and the amounts of erk5 , bad , and bim transcripts were measured by RT PCR. b , c , Homozygous flox SCG neurons were transiently transfected with a Bad or a Bim reporter luciferase plasmid and a pRL-Tk plasmid 20 h or 2 h prior to being infected with an adenovirus encoding GFP or Cre for 18 h and 36 h, respectively. The transcriptional activity was measured by the Dual-Luciferase reporter assay system. d - f , PC6.3 cells were transiently co-transfected with a Bad or a Bim reporter luciferase plasmid and a pRL-Tk plasmid together with (+) or without (−) an expression vector encoding flag-tagged DN-ERK5. The following day the cells were cultured in differentiating medium without or with UO126 (10 μM) or wortmanin (50 nM), for a further 36 hours. The inhibitors were replaced every 12 h. Cell extracts were analyzed for the expression of DN-ERK5, Bad and Bim, and for the phosphorylation (P) of ERK5 by immunoblot ( d ). The detection of tubulin expression was performed to monitor protein loading. The transcriptional activity was measured by the Dual-Luciferase reporter assay system ( e , f ). All the data correspond to the mean ± SE of three independent experiments performed in duplicate.
    Figure Legend Snippet: ERK5 regulates Bad and Bim transcription. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. Total RNA was extracted and the amounts of erk5 , bad , and bim transcripts were measured by RT PCR. b , c , Homozygous flox SCG neurons were transiently transfected with a Bad or a Bim reporter luciferase plasmid and a pRL-Tk plasmid 20 h or 2 h prior to being infected with an adenovirus encoding GFP or Cre for 18 h and 36 h, respectively. The transcriptional activity was measured by the Dual-Luciferase reporter assay system. d - f , PC6.3 cells were transiently co-transfected with a Bad or a Bim reporter luciferase plasmid and a pRL-Tk plasmid together with (+) or without (−) an expression vector encoding flag-tagged DN-ERK5. The following day the cells were cultured in differentiating medium without or with UO126 (10 μM) or wortmanin (50 nM), for a further 36 hours. The inhibitors were replaced every 12 h. Cell extracts were analyzed for the expression of DN-ERK5, Bad and Bim, and for the phosphorylation (P) of ERK5 by immunoblot ( d ). The detection of tubulin expression was performed to monitor protein loading. The transcriptional activity was measured by the Dual-Luciferase reporter assay system ( e , f ). All the data correspond to the mean ± SE of three independent experiments performed in duplicate.

    Techniques Used: Infection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Reporter Assay, Expressing

    ERK5 regulates Foxo3a-dependent transcription of bim via PKB. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. b , c , PC6.3 cells were transiently transfected with (+) or without (−) DN-ERK5. The following day the cells were cultured in differentiating medium containing NGF for a further 36 hours. Extracts were analyzed for the expression and the phosphorylation (P) of PKB at Thr308 or at Ser473 by immunoblot ( a , b ). Chromatin was immunoprecipitated with an antibody to Foxo3a. The precipitated DNA was amplified by semi-quantitative PCR ( c ). Input DNA levels were used to monitor transfection efficiency. Similar results were obtained in two independent experiments.
    Figure Legend Snippet: ERK5 regulates Foxo3a-dependent transcription of bim via PKB. a , Homozygous flox SCG neurons were infected with an adenovirus encoding GFP or Cre. The cells were cultured in presence of NGF (50 ng/ml) for the indicated times. b , c , PC6.3 cells were transiently transfected with (+) or without (−) DN-ERK5. The following day the cells were cultured in differentiating medium containing NGF for a further 36 hours. Extracts were analyzed for the expression and the phosphorylation (P) of PKB at Thr308 or at Ser473 by immunoblot ( a , b ). Chromatin was immunoprecipitated with an antibody to Foxo3a. The precipitated DNA was amplified by semi-quantitative PCR ( c ). Input DNA levels were used to monitor transfection efficiency. Similar results were obtained in two independent experiments.

    Techniques Used: Infection, Cell Culture, Transfection, Expressing, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    erk5 gene deletion sensitizes neurons to apoptosis. Homozygous flox ( a - g ) or wild type ( h ) SCG neurons were not infected or infected with a control adenovirus (GFP) or with an adenovirus encoding Cre at 100 MOI. The cells were cultured for a further 36 h ( a ) or 48 h ( b , d - h ) in presence of NGF (50 ng/ml), unless indicated otherwise. a, Immunofluorescence analysis of SCG neurons to detect GFP (green) and Cre (anti-Cre antibody, red) expression demonstrates that 100% of the cells were infected by the recombinant adenoviruses. DNA was stained with DAPI (blue). Scale bar, 25 μM. b , (i) Genomic DNA isolated from the cells was amplified by PCR with primers specific for the erk5 gene. erk5f and erk5- correspond to the erk5 - flox and disrupted allele, respectively; (ii) Proteins were extracted and analyzed by immunoblot using specific antibodies to ERK5 and to tubulin. c , Extracts were analyzed for ERK5 expression by immunoblot. The detection of tubulin expression was performed to monitor protein loading. Where indicated, NGF was removed (− NGF) for 15 min and 30 min and re-added (+ NGF) for 30 min, prior to the cells being harvested. CIP treatment of the extract prior to analysis is indicated. Similar results were obtained in two independent experiments. d , Extracts were analyzed for JNK expression and phosphorylation (P) by immunoblot. Where indicated, NGF was removed (− NGF) for 6 h prior to the cells being harvested. Similar results were obtained in two independent experiments. e , Phase-contrast photomicrographs of representative fields of SCG neurons expressing GFP or Cre are shown. Scale bar, 25 μM. f , SCG neurons were incubated with Hoechst 33342 and propidium iodide to distinguish viable, necrotic and apoptotic cells. Only neurons that had clearly segmented and condensed chromatin were counted as apoptotic. The classification criteria are shown in supplementary Figure 1 . g , h , Caspase 3 activity was measured by caspase assay. In some experiments, NGF was removed (0) for 18 h prior to the cells being harvested ( f, g ). The data, expressed as the mean +/− standard error (SE), were generated from three independent experiments performed in duplicate ( f-h ). *, P ≤ 0.001 indicates a significant difference between GFP and Cre infected neurons. The electrophoretic mobility shift caused by the phosphorylation of ERK5 is indicated by an arrow.
    Figure Legend Snippet: erk5 gene deletion sensitizes neurons to apoptosis. Homozygous flox ( a - g ) or wild type ( h ) SCG neurons were not infected or infected with a control adenovirus (GFP) or with an adenovirus encoding Cre at 100 MOI. The cells were cultured for a further 36 h ( a ) or 48 h ( b , d - h ) in presence of NGF (50 ng/ml), unless indicated otherwise. a, Immunofluorescence analysis of SCG neurons to detect GFP (green) and Cre (anti-Cre antibody, red) expression demonstrates that 100% of the cells were infected by the recombinant adenoviruses. DNA was stained with DAPI (blue). Scale bar, 25 μM. b , (i) Genomic DNA isolated from the cells was amplified by PCR with primers specific for the erk5 gene. erk5f and erk5- correspond to the erk5 - flox and disrupted allele, respectively; (ii) Proteins were extracted and analyzed by immunoblot using specific antibodies to ERK5 and to tubulin. c , Extracts were analyzed for ERK5 expression by immunoblot. The detection of tubulin expression was performed to monitor protein loading. Where indicated, NGF was removed (− NGF) for 15 min and 30 min and re-added (+ NGF) for 30 min, prior to the cells being harvested. CIP treatment of the extract prior to analysis is indicated. Similar results were obtained in two independent experiments. d , Extracts were analyzed for JNK expression and phosphorylation (P) by immunoblot. Where indicated, NGF was removed (− NGF) for 6 h prior to the cells being harvested. Similar results were obtained in two independent experiments. e , Phase-contrast photomicrographs of representative fields of SCG neurons expressing GFP or Cre are shown. Scale bar, 25 μM. f , SCG neurons were incubated with Hoechst 33342 and propidium iodide to distinguish viable, necrotic and apoptotic cells. Only neurons that had clearly segmented and condensed chromatin were counted as apoptotic. The classification criteria are shown in supplementary Figure 1 . g , h , Caspase 3 activity was measured by caspase assay. In some experiments, NGF was removed (0) for 18 h prior to the cells being harvested ( f, g ). The data, expressed as the mean +/− standard error (SE), were generated from three independent experiments performed in duplicate ( f-h ). *, P ≤ 0.001 indicates a significant difference between GFP and Cre infected neurons. The electrophoretic mobility shift caused by the phosphorylation of ERK5 is indicated by an arrow.

    Techniques Used: Infection, Cell Culture, Immunofluorescence, Expressing, Recombinant, Staining, Isolation, Amplification, Polymerase Chain Reaction, Incubation, Activity Assay, Caspase Assay, Generated, Electrophoretic Mobility Shift Assay

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    SgG2 interacts with <t>NGF.</t> (A) Sensorgram showing the interaction of SgG2 with increasing concentrations of <t>NGF.</t> The concentrations of NGF are indicated at the right side of the sensorgram. (B) Sensorgram showing the interaction of SgG2 with NGF and the lack of interaction with TNF-α, IFN-α and IL-1. All analytes were injected at a 100 nM concentration. (C) Sensorgram depicting the interaction between increasing concentrations of SgG2 and NGF coupled on a sensor chip. As a negative control we used the same concentrations of purified HSV-2 gD. (D) Saturation curve for the binding of NGF to SgG2. The derived KD is shown (E) Sensorgram showing the interaction of coupled SgG2 with CXCL12β and NGF injected alone or in combination. In all cases the arrow indicates the end of injection. Abbreviations: Diff. Resp., Differential response; M, molar; R.U., response units; s, seconds.
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    Image Search Results


    Suggested model. In order to arrive at the cell body and subsequently the CNS, rabies virus hijacks a fast route using the p75NTR endosomal pathway. In a p75NTR dependent path, RABV manipulates the axonal transport machinery to migrate faster to the cell body. An alternative, slower path, may involve alternative RABV receptors.

    Journal: PLoS Pathogens

    Article Title: Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

    doi: 10.1371/journal.ppat.1004348

    Figure Lengend Snippet: Suggested model. In order to arrive at the cell body and subsequently the CNS, rabies virus hijacks a fast route using the p75NTR endosomal pathway. In a p75NTR dependent path, RABV manipulates the axonal transport machinery to migrate faster to the cell body. An alternative, slower path, may involve alternative RABV receptors.

    Article Snippet: For RABV-p75 imaging, explant cultures were incubated with fluorescent anti-p75NTR (ANT-007-AO, Alomone Labs) for 10 minutes and washed 3 times in poor neurobasal medium prior to imaging.

    Techniques:

    RABV is retrogradely transported with neurotrophin receptors. ( A–D ), Retrograde transport of EGFP-RABV, added to the distal axon compartment of DRG explant previously treated with fluorescent antibodies against p75NTR and TrkA. Arrowheads: RABV puncta positive for p75NTR, arrows: RABV puncta positive for p75NTR and TrkA. Scale bar = 10 µm. ( E,F ) Co-localisation of RABV with p75NTR and TrkA calculated from two and one experiments, respectively. ( G ) Trajectories of RABV trafficked with neurotrophin receptors (NTFR, Blue) or without (Red), illustrating a more processive displacement over time of RABV with NTFR. ( H ) Merged kymographs of RABV (green) p75NTR (red) and TrkA (cyan), drawn for multi-channel time lapse. Vertical scale bar = 5 µm, horizontal scale bar = 40 seconds.

    Journal: PLoS Pathogens

    Article Title: Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

    doi: 10.1371/journal.ppat.1004348

    Figure Lengend Snippet: RABV is retrogradely transported with neurotrophin receptors. ( A–D ), Retrograde transport of EGFP-RABV, added to the distal axon compartment of DRG explant previously treated with fluorescent antibodies against p75NTR and TrkA. Arrowheads: RABV puncta positive for p75NTR, arrows: RABV puncta positive for p75NTR and TrkA. Scale bar = 10 µm. ( E,F ) Co-localisation of RABV with p75NTR and TrkA calculated from two and one experiments, respectively. ( G ) Trajectories of RABV trafficked with neurotrophin receptors (NTFR, Blue) or without (Red), illustrating a more processive displacement over time of RABV with NTFR. ( H ) Merged kymographs of RABV (green) p75NTR (red) and TrkA (cyan), drawn for multi-channel time lapse. Vertical scale bar = 5 µm, horizontal scale bar = 40 seconds.

    Article Snippet: For RABV-p75 imaging, explant cultures were incubated with fluorescent anti-p75NTR (ANT-007-AO, Alomone Labs) for 10 minutes and washed 3 times in poor neurobasal medium prior to imaging.

    Techniques:

    RABV binds and internalizes with p75NTR in DRG neuron tips. Co-localization of EGFP-RABV with p75NTR is shown by live TIRF imaging and sub-pixel localization algorithms. ( A ) RABV-p75 particles shift from the periphery to the center of the growth cone, where they are internalized into the cell. Lower panels zoom in on dashed square, showing co-localized puncta (left) shifting towards the center of the growth cone (middle) until finally internalized (right). ( B ) Presentation of six separate events of RABV and p75NTR binding and internalization on the surface of the growth cone shown in (A). Colored trajectories denote displacement from point of detection to point of disappearance. ( C ) RABV and p75NTR are internalized together, illustrated by corresponding plots of puncta intensity over time (normalized to background), calculated for co-localized particles shown in lower panels of (A). Scale bars = 5 µm. ( D ) Zoom-in on colocalized RABV and p75 spot, taken from panel (A), scale bar = 1 µm. ( E ) Overlay of 1D-Gaussian fits of p75 and RABV intensity profiles at the x-axis of the image in panel (D). ( F ) Representative overlay of radial symmetry fits of the x-y intensity profiles of p75 and RABV spots. σ is the standard deviation of each fitting function; distance between the two spot centers is 51.3 nm. ( G ) Knockdown of p75NTR decreases rabies virus infection for shorts time incubation. DRGs embryonic cells infected with lentiviral vectors (LV) containing 4 different EGFP-tagged shRNA's against p75NTR or LV-EGFP, were transfected with RABV for 30 or 120 minutes. Low levels of infected neurons were found in shRNA-p75-EGFP cells Average RABV infection rates were normalized to LV-EGFP controls (n = 4 experiments, error bars = SEM, *p

    Journal: PLoS Pathogens

    Article Title: Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

    doi: 10.1371/journal.ppat.1004348

    Figure Lengend Snippet: RABV binds and internalizes with p75NTR in DRG neuron tips. Co-localization of EGFP-RABV with p75NTR is shown by live TIRF imaging and sub-pixel localization algorithms. ( A ) RABV-p75 particles shift from the periphery to the center of the growth cone, where they are internalized into the cell. Lower panels zoom in on dashed square, showing co-localized puncta (left) shifting towards the center of the growth cone (middle) until finally internalized (right). ( B ) Presentation of six separate events of RABV and p75NTR binding and internalization on the surface of the growth cone shown in (A). Colored trajectories denote displacement from point of detection to point of disappearance. ( C ) RABV and p75NTR are internalized together, illustrated by corresponding plots of puncta intensity over time (normalized to background), calculated for co-localized particles shown in lower panels of (A). Scale bars = 5 µm. ( D ) Zoom-in on colocalized RABV and p75 spot, taken from panel (A), scale bar = 1 µm. ( E ) Overlay of 1D-Gaussian fits of p75 and RABV intensity profiles at the x-axis of the image in panel (D). ( F ) Representative overlay of radial symmetry fits of the x-y intensity profiles of p75 and RABV spots. σ is the standard deviation of each fitting function; distance between the two spot centers is 51.3 nm. ( G ) Knockdown of p75NTR decreases rabies virus infection for shorts time incubation. DRGs embryonic cells infected with lentiviral vectors (LV) containing 4 different EGFP-tagged shRNA's against p75NTR or LV-EGFP, were transfected with RABV for 30 or 120 minutes. Low levels of infected neurons were found in shRNA-p75-EGFP cells Average RABV infection rates were normalized to LV-EGFP controls (n = 4 experiments, error bars = SEM, *p

    Article Snippet: For RABV-p75 imaging, explant cultures were incubated with fluorescent anti-p75NTR (ANT-007-AO, Alomone Labs) for 10 minutes and washed 3 times in poor neurobasal medium prior to imaging.

    Techniques: Imaging, Binding Assay, Standard Deviation, Infection, Incubation, shRNA, Transfection

    RABV travels faster and is more directed when transported with p75NTR. ( A–C ) Multi-channel live imaging of EGFP-RABV 2 hours after addition to distal axon compartment of DRG explant previously treated with a fluorescent antibody against p75NTR. Arrowheads: p75NTR-positive RABV puncta, scale bar = 10 µm. ( D,E ) Kymographs of and P75NTR extracted from time lapse depicted in (A–C). ( F ) RABV-only tracks (green) are less directed than RABV-p75NTR tracks (yellow), as shown when overlaying corresponding kymographs. Vertical scale bar = 5 µm, horizontal scale bar = 40 seconds. ( G–O ) Characterization of directed RABV puncta, transported with and without p75NTR, n = 184 and n = 122, respectively. (G) RABV presents higher speeds when transported with p75NTR, due to less frequent (H) and shorter pauses (I). Overall RABV-p75NTR spent less time paused on average (J), Diameter and intensity measurements revealed that RABV puncta positive for p75NTR were larger ( K ) and had higher intensity levels ( L ) than p75NTR-negative puncta. ( M–O ) p75NTR positive puncta (blue) are faster, more directed and present higher displacements over time, compared to p75NTR negative puncta (red), illustrated by distribution of instantaneous velocities in (M) (RABV+p75: n = 8051 events; RABV-p75: n = 7423 events) displacement plotted over time (N) and mean square displacement (O). Data is pulled from two separate experiments, error bars represent SEM. *p

    Journal: PLoS Pathogens

    Article Title: Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

    doi: 10.1371/journal.ppat.1004348

    Figure Lengend Snippet: RABV travels faster and is more directed when transported with p75NTR. ( A–C ) Multi-channel live imaging of EGFP-RABV 2 hours after addition to distal axon compartment of DRG explant previously treated with a fluorescent antibody against p75NTR. Arrowheads: p75NTR-positive RABV puncta, scale bar = 10 µm. ( D,E ) Kymographs of and P75NTR extracted from time lapse depicted in (A–C). ( F ) RABV-only tracks (green) are less directed than RABV-p75NTR tracks (yellow), as shown when overlaying corresponding kymographs. Vertical scale bar = 5 µm, horizontal scale bar = 40 seconds. ( G–O ) Characterization of directed RABV puncta, transported with and without p75NTR, n = 184 and n = 122, respectively. (G) RABV presents higher speeds when transported with p75NTR, due to less frequent (H) and shorter pauses (I). Overall RABV-p75NTR spent less time paused on average (J), Diameter and intensity measurements revealed that RABV puncta positive for p75NTR were larger ( K ) and had higher intensity levels ( L ) than p75NTR-negative puncta. ( M–O ) p75NTR positive puncta (blue) are faster, more directed and present higher displacements over time, compared to p75NTR negative puncta (red), illustrated by distribution of instantaneous velocities in (M) (RABV+p75: n = 8051 events; RABV-p75: n = 7423 events) displacement plotted over time (N) and mean square displacement (O). Data is pulled from two separate experiments, error bars represent SEM. *p

    Article Snippet: For RABV-p75 imaging, explant cultures were incubated with fluorescent anti-p75NTR (ANT-007-AO, Alomone Labs) for 10 minutes and washed 3 times in poor neurobasal medium prior to imaging.

    Techniques: Imaging

    TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells through its type I receptor in a dose-dependent manner. After 24-h culture in growth medium, SCDC2 cells were starved for 24 h. The starved cells were then treated with (A) TGF-β1 at various concentrations for 24 h, or (B) pretreated with or without TGF-β type I receptor inhibitor SB-431542 (10 µ M) for 30 min and then with or without TGF-β1 (10 ng/ml) for 24 h. (C) Starved cells were treated with or without TGF-β1 (10 ng/ml) for the indicated times. The relative expression level of NGF was evaluated using reverse transcription-quantitative polymerase chain reaction. Data represent the mean ± standard deviation (n=6). * P

    Journal: International Journal of Molecular Medicine

    Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

    doi: 10.3892/ijmm_2018.3714

    Figure Lengend Snippet: TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells through its type I receptor in a dose-dependent manner. After 24-h culture in growth medium, SCDC2 cells were starved for 24 h. The starved cells were then treated with (A) TGF-β1 at various concentrations for 24 h, or (B) pretreated with or without TGF-β type I receptor inhibitor SB-431542 (10 µ M) for 30 min and then with or without TGF-β1 (10 ng/ml) for 24 h. (C) Starved cells were treated with or without TGF-β1 (10 ng/ml) for the indicated times. The relative expression level of NGF was evaluated using reverse transcription-quantitative polymerase chain reaction. Data represent the mean ± standard deviation (n=6). * P

    Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    IL-1β and TNF-α suppressed the TGF-β1-induced mRNA expression of NGF in SCDC2 cells by abrogating Smad2/3 and p38 MAPK activities. The effects of IL-1β and TNF-α on TGF-β1-induced mRNA expression of NGF in SCDC2 cells were evaluated using RT-qPCR. The cells were treated with or without (A) IL-1β alone or (B) TNF-α alone at indicated concentrations, (C) TGF-β1 (10 ng/ml) and/or IL-1β (10 ng/ml), and (D) TGF-β1 (10 ng/ml) and/or TNF-α (10 ng/ml). Data represent the mean ± standard deviation (n=6). * P

    Journal: International Journal of Molecular Medicine

    Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

    doi: 10.3892/ijmm_2018.3714

    Figure Lengend Snippet: IL-1β and TNF-α suppressed the TGF-β1-induced mRNA expression of NGF in SCDC2 cells by abrogating Smad2/3 and p38 MAPK activities. The effects of IL-1β and TNF-α on TGF-β1-induced mRNA expression of NGF in SCDC2 cells were evaluated using RT-qPCR. The cells were treated with or without (A) IL-1β alone or (B) TNF-α alone at indicated concentrations, (C) TGF-β1 (10 ng/ml) and/or IL-1β (10 ng/ml), and (D) TGF-β1 (10 ng/ml) and/or TNF-α (10 ng/ml). Data represent the mean ± standard deviation (n=6). * P

    Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

    TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells in Smad2/3-dependent and p38 MAPK-dependent manners. Effects of (A) SIS3 (10 µ M), and (B) SB203580 (10 µ M) on expression of NGF mRNA were evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). * P

    Journal: International Journal of Molecular Medicine

    Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

    doi: 10.3892/ijmm_2018.3714

    Figure Lengend Snippet: TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells in Smad2/3-dependent and p38 MAPK-dependent manners. Effects of (A) SIS3 (10 µ M), and (B) SB203580 (10 µ M) on expression of NGF mRNA were evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). * P

    Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

    Techniques: Expressing, Standard Deviation

    Process of differentiation of H-iris iPS cells to nervous system cells. Cells were pre-differentiated from ( a ) H-iris iPS cells to ( b ) neural stem/progenitor cells by adhesive culture. ( c ) Cells sorted with p75NTR were differentiated into neurons. By changing the medium condition, the nerve cells were differentiated into Recoverin-positive cells. ( d ) In contrast, after suspension culture, neurites were elongated by adhesive culture and differentiated into retinal ganglion cells. Bars: 100 μm.

    Journal: Cells

    Article Title: Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells

    doi: 10.3390/cells10040743

    Figure Lengend Snippet: Process of differentiation of H-iris iPS cells to nervous system cells. Cells were pre-differentiated from ( a ) H-iris iPS cells to ( b ) neural stem/progenitor cells by adhesive culture. ( c ) Cells sorted with p75NTR were differentiated into neurons. By changing the medium condition, the nerve cells were differentiated into Recoverin-positive cells. ( d ) In contrast, after suspension culture, neurites were elongated by adhesive culture and differentiated into retinal ganglion cells. Bars: 100 μm.

    Article Snippet: Paraffin sections were prepared from the fixed human iris tissue in the usual manner and incubated with anti-p75NTR polyclonal antibody (1:200; Alomone Labs, Jerusalem, Israel) for 1 h at 37 °C.

    Techniques:

    Cells sorted with p75NTR can concentrate Recoverin-positive cells. ( a ) Before p75NTR sorting, cells with large cytoplasms ( arrowheads ) were mixed. ( b ) Fixed cells were analyzed for p75NTR and Nestin by FCM. Most of the cells sorted in the strong positive region of p75NTR were double-positive for ( c ) p75NTR and ( d ) Nestin. ( e , f ) The morphology of cells sorted by their strong positivity for ( e ) p75NTR or ( f ) their weak positivity or negativity for p75NTR. ( g ) Relative semi-quantitative analysis of Recoverin gene expression in cells selected by p75NTR, and pre/post-differentiated cells. ( h ) Fluorescent immunostaining of Recoverin in differentiated cells. Bar in panel ( a ): 50 μm, bars in panels ( c , d ): 20 μm, bars in panels ( e , f , h ): 100 μm.

    Journal: Cells

    Article Title: Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells

    doi: 10.3390/cells10040743

    Figure Lengend Snippet: Cells sorted with p75NTR can concentrate Recoverin-positive cells. ( a ) Before p75NTR sorting, cells with large cytoplasms ( arrowheads ) were mixed. ( b ) Fixed cells were analyzed for p75NTR and Nestin by FCM. Most of the cells sorted in the strong positive region of p75NTR were double-positive for ( c ) p75NTR and ( d ) Nestin. ( e , f ) The morphology of cells sorted by their strong positivity for ( e ) p75NTR or ( f ) their weak positivity or negativity for p75NTR. ( g ) Relative semi-quantitative analysis of Recoverin gene expression in cells selected by p75NTR, and pre/post-differentiated cells. ( h ) Fluorescent immunostaining of Recoverin in differentiated cells. Bar in panel ( a ): 50 μm, bars in panels ( c , d ): 20 μm, bars in panels ( e , f , h ): 100 μm.

    Article Snippet: Paraffin sections were prepared from the fixed human iris tissue in the usual manner and incubated with anti-p75NTR polyclonal antibody (1:200; Alomone Labs, Jerusalem, Israel) for 1 h at 37 °C.

    Techniques: Expressing, Immunostaining

    p75NTR-positive cells observed in human iris tissue and cultured cells: ( a ) HE-stained human iris tissue; * The lens side of the iris. ( b ) Differential interference contrast image of iris tissue. ( c ) Fluorescent immunostaining of p75NTR; arrowheads = p75NTR-positive cells. ( d ) Iris-derived cells were cultured for 10 days; the inset shows cells growing from around the pigmented cells on the third day of culture. ( e ) p75NTR-positive cells (region of Gate 1, G1) were isolated with a cell sorter; the blue line represents the histogram of negative cells. ( f ) Morphology of p75NTR-positive sorted cells. Bars in panels ( a – c ), 50 μm; bars in panel ( d )’s inset and panel ( f ), 100 μm; bar in panel ( d ), 200 μm.

    Journal: Cells

    Article Title: Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells

    doi: 10.3390/cells10040743

    Figure Lengend Snippet: p75NTR-positive cells observed in human iris tissue and cultured cells: ( a ) HE-stained human iris tissue; * The lens side of the iris. ( b ) Differential interference contrast image of iris tissue. ( c ) Fluorescent immunostaining of p75NTR; arrowheads = p75NTR-positive cells. ( d ) Iris-derived cells were cultured for 10 days; the inset shows cells growing from around the pigmented cells on the third day of culture. ( e ) p75NTR-positive cells (region of Gate 1, G1) were isolated with a cell sorter; the blue line represents the histogram of negative cells. ( f ) Morphology of p75NTR-positive sorted cells. Bars in panels ( a – c ), 50 μm; bars in panel ( d )’s inset and panel ( f ), 100 μm; bar in panel ( d ), 200 μm.

    Article Snippet: Paraffin sections were prepared from the fixed human iris tissue in the usual manner and incubated with anti-p75NTR polyclonal antibody (1:200; Alomone Labs, Jerusalem, Israel) for 1 h at 37 °C.

    Techniques: Cell Culture, Staining, Immunostaining, Derivative Assay, Isolation

    SgG2 interacts with NGF. (A) Sensorgram showing the interaction of SgG2 with increasing concentrations of NGF. The concentrations of NGF are indicated at the right side of the sensorgram. (B) Sensorgram showing the interaction of SgG2 with NGF and the lack of interaction with TNF-α, IFN-α and IL-1. All analytes were injected at a 100 nM concentration. (C) Sensorgram depicting the interaction between increasing concentrations of SgG2 and NGF coupled on a sensor chip. As a negative control we used the same concentrations of purified HSV-2 gD. (D) Saturation curve for the binding of NGF to SgG2. The derived KD is shown (E) Sensorgram showing the interaction of coupled SgG2 with CXCL12β and NGF injected alone or in combination. In all cases the arrow indicates the end of injection. Abbreviations: Diff. Resp., Differential response; M, molar; R.U., response units; s, seconds.

    Journal: PLoS Pathogens

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    doi: 10.1371/journal.ppat.1004571

    Figure Lengend Snippet: SgG2 interacts with NGF. (A) Sensorgram showing the interaction of SgG2 with increasing concentrations of NGF. The concentrations of NGF are indicated at the right side of the sensorgram. (B) Sensorgram showing the interaction of SgG2 with NGF and the lack of interaction with TNF-α, IFN-α and IL-1. All analytes were injected at a 100 nM concentration. (C) Sensorgram depicting the interaction between increasing concentrations of SgG2 and NGF coupled on a sensor chip. As a negative control we used the same concentrations of purified HSV-2 gD. (D) Saturation curve for the binding of NGF to SgG2. The derived KD is shown (E) Sensorgram showing the interaction of coupled SgG2 with CXCL12β and NGF injected alone or in combination. In all cases the arrow indicates the end of injection. Abbreviations: Diff. Resp., Differential response; M, molar; R.U., response units; s, seconds.

    Article Snippet: In brief, 340 μL of rat tail collagen I (BD Biosciences, San Jose, CA) were mixed with 40 μL of 10x MEM, 10 μL of HEPES or the vCKBPs at a final concentration of 50 nM, and mouse NGF 2.5S (N-100, Alomone labs, Jerusalem, Israel) at a final concentration of 0.25 nM.

    Techniques: Injection, Concentration Assay, Chromatin Immunoprecipitation, Negative Control, Purification, Binding Assay, Derivative Assay

    SgG2 reduces NGF induced TrkA internalization and retrograde transport. SCG dissociated neurons were grown during 5 DIV and deprived of NGF for 16 h. (A) Neurons were stimulated with NGF alone or together with the indicated viral proteins during 0, 15 and 120 min. TrkA internalization was analyzed by immunofluorescence without permeabilization and normalized for F-actin using phalloidin staining. Solid arrowhead points to a non-internalized cluster of TrkA at the plasma membrane. Images show a projection of at least 3 image planes. All images correspond to the same experiment, representative of two independent experiments ( n = 12 for each condition). Scale bar represents 5 μm. The graph shows the quantification of the TrkA/phalloidin staining at the plasma membrane over time in samples treated with NGF plus the indicated protein or buffer. (B) Mouse SCG dissociated neurons were grown during 7 DIV using microfluidic devices. Neurons were deprived of NGF for 16 h and NGF and viral proteins were added to the distal axon compartment during 120 min. TrkA phosphorylation was analyzed by immunofluorescence after permeabilization with Triton X-100 and normalized using phalloidin. The images and the graph correspond to the same experiment and are representative of three independent experiments. Scale bar 10 μm. n = 10 fields for each condition. A two-tailed unpaired T-test was used to calculate significance in (A) and (B) . *** P

    Journal: PLoS Pathogens

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    doi: 10.1371/journal.ppat.1004571

    Figure Lengend Snippet: SgG2 reduces NGF induced TrkA internalization and retrograde transport. SCG dissociated neurons were grown during 5 DIV and deprived of NGF for 16 h. (A) Neurons were stimulated with NGF alone or together with the indicated viral proteins during 0, 15 and 120 min. TrkA internalization was analyzed by immunofluorescence without permeabilization and normalized for F-actin using phalloidin staining. Solid arrowhead points to a non-internalized cluster of TrkA at the plasma membrane. Images show a projection of at least 3 image planes. All images correspond to the same experiment, representative of two independent experiments ( n = 12 for each condition). Scale bar represents 5 μm. The graph shows the quantification of the TrkA/phalloidin staining at the plasma membrane over time in samples treated with NGF plus the indicated protein or buffer. (B) Mouse SCG dissociated neurons were grown during 7 DIV using microfluidic devices. Neurons were deprived of NGF for 16 h and NGF and viral proteins were added to the distal axon compartment during 120 min. TrkA phosphorylation was analyzed by immunofluorescence after permeabilization with Triton X-100 and normalized using phalloidin. The images and the graph correspond to the same experiment and are representative of three independent experiments. Scale bar 10 μm. n = 10 fields for each condition. A two-tailed unpaired T-test was used to calculate significance in (A) and (B) . *** P

    Article Snippet: In brief, 340 μL of rat tail collagen I (BD Biosciences, San Jose, CA) were mixed with 40 μL of 10x MEM, 10 μL of HEPES or the vCKBPs at a final concentration of 50 nM, and mouse NGF 2.5S (N-100, Alomone labs, Jerusalem, Israel) at a final concentration of 0.25 nM.

    Techniques: Immunofluorescence, Staining, Two Tailed Test

    SgG2 modifies NGF-TrkA signaling. Mouse SCG dissociated neurons were grown during 5 days in vitro (DIV). (A) Neurons were deprived of NGF for 16 h and were stimulated with NGF alone, NGF plus SgG2 or SgG2 alone during 5 min. TrkA-SgG2 interaction was analyzed by TrkA immunoprecipitation (IP) followed by Western blot (WB) to detect SgG2. Molecular sizes in kDa and the position of SgG2 (empty arrowhead) and TrkA (solid arrowhead) are indicated. The experiment shown is representative of three independent assays. (B-F) Neurons were deprived of NGF for 16 h and were stimulated with NGF and the indicated viral proteins during 0, 15 and 120 min. (B) The phosphorylation levels of TrkA, ERK, AKT and cofilin were analyzed by Western blot (WB) using specific antibodies. Detection of actin was used as a loading control. All blots correspond to the same experiment. Graphs show statistical analysis for (C) p-TrkA, (D) p-ERK, (E) pAKT and (F) p-cofilin levels. p-TrkA n = 4; p-ERK n = 4; p-AKT n = 4; p-cofilin n = 3. To calculate significance a two-tailed unpaired T-test was employed; * P

    Journal: PLoS Pathogens

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    doi: 10.1371/journal.ppat.1004571

    Figure Lengend Snippet: SgG2 modifies NGF-TrkA signaling. Mouse SCG dissociated neurons were grown during 5 days in vitro (DIV). (A) Neurons were deprived of NGF for 16 h and were stimulated with NGF alone, NGF plus SgG2 or SgG2 alone during 5 min. TrkA-SgG2 interaction was analyzed by TrkA immunoprecipitation (IP) followed by Western blot (WB) to detect SgG2. Molecular sizes in kDa and the position of SgG2 (empty arrowhead) and TrkA (solid arrowhead) are indicated. The experiment shown is representative of three independent assays. (B-F) Neurons were deprived of NGF for 16 h and were stimulated with NGF and the indicated viral proteins during 0, 15 and 120 min. (B) The phosphorylation levels of TrkA, ERK, AKT and cofilin were analyzed by Western blot (WB) using specific antibodies. Detection of actin was used as a loading control. All blots correspond to the same experiment. Graphs show statistical analysis for (C) p-TrkA, (D) p-ERK, (E) pAKT and (F) p-cofilin levels. p-TrkA n = 4; p-ERK n = 4; p-AKT n = 4; p-cofilin n = 3. To calculate significance a two-tailed unpaired T-test was employed; * P

    Article Snippet: In brief, 340 μL of rat tail collagen I (BD Biosciences, San Jose, CA) were mixed with 40 μL of 10x MEM, 10 μL of HEPES or the vCKBPs at a final concentration of 50 nM, and mouse NGF 2.5S (N-100, Alomone labs, Jerusalem, Israel) at a final concentration of 0.25 nM.

    Techniques: In Vitro, Immunoprecipitation, Western Blot, Two Tailed Test

    SgG2 increases NGF-dependent axonal growth. (A,B,D) Mouse SCGs were grown as explants in collagen matrix in the presence of the indicated trophic factors and viral proteins. Neurons were stained with Tuj1 antibody targeting class III ß–tubulin. Nuclei were stained with TO-PRO-3. Images are a projection of at least 3 stacks, correspond to the same experiment, and are representative of three (A,D) or two (B) independent experiments. The graphs below represent the quantification from three (A,D) or two (B) independent experiments. To calculate significance, a two-tailed unpaired T-test was applied. *** P

    Journal: PLoS Pathogens

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    doi: 10.1371/journal.ppat.1004571

    Figure Lengend Snippet: SgG2 increases NGF-dependent axonal growth. (A,B,D) Mouse SCGs were grown as explants in collagen matrix in the presence of the indicated trophic factors and viral proteins. Neurons were stained with Tuj1 antibody targeting class III ß–tubulin. Nuclei were stained with TO-PRO-3. Images are a projection of at least 3 stacks, correspond to the same experiment, and are representative of three (A,D) or two (B) independent experiments. The graphs below represent the quantification from three (A,D) or two (B) independent experiments. To calculate significance, a two-tailed unpaired T-test was applied. *** P

    Article Snippet: In brief, 340 μL of rat tail collagen I (BD Biosciences, San Jose, CA) were mixed with 40 μL of 10x MEM, 10 μL of HEPES or the vCKBPs at a final concentration of 50 nM, and mouse NGF 2.5S (N-100, Alomone labs, Jerusalem, Israel) at a final concentration of 0.25 nM.

    Techniques: Staining, Two Tailed Test

    SgG2 promotes the incorporation of TrkA into different microdomains of the plasma membrane. Mouse SCG dissociated neurons were grown during 5 DIV. Neurons were deprived of NGF for 16 h and subsequently stimulated with NGF, vCKBPs or both. Colocalization between TrkA and two different subtypes of lipid rafts was studied using immunofluorescence without permeabilization. TrkA colocalization with GM1 rafts 2 min (A) and 10 min (B) post-stimulation. TrkA colocalization with GM3 rafts 2 min (C) , and 10 min (D) post-stimulation. Confocal microscopy images correspond to one representative cell from each condition. The +ves image displays pseudocolored pixels from the areas within the plasma membrane in which both TrkA and the corresponding subtype of lipid rafts pixel value exceed the mean. Scale bar represents 5 μm. Graphs show the average values (mean±SEM) obtained for PC and ICQ. Plots in (A) represent n = 18–26 neurons from three independent assays. Plots in (B) show the results obtained for 15 cells from two independent experiments. Plots in (C) represent n = 21–35 neurons from three independent assays. Plots in (D) represent 16–23 neurons from two independent assays. Two-tailed unpaired T-test, * P

    Journal: PLoS Pathogens

    Article Title: Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    doi: 10.1371/journal.ppat.1004571

    Figure Lengend Snippet: SgG2 promotes the incorporation of TrkA into different microdomains of the plasma membrane. Mouse SCG dissociated neurons were grown during 5 DIV. Neurons were deprived of NGF for 16 h and subsequently stimulated with NGF, vCKBPs or both. Colocalization between TrkA and two different subtypes of lipid rafts was studied using immunofluorescence without permeabilization. TrkA colocalization with GM1 rafts 2 min (A) and 10 min (B) post-stimulation. TrkA colocalization with GM3 rafts 2 min (C) , and 10 min (D) post-stimulation. Confocal microscopy images correspond to one representative cell from each condition. The +ves image displays pseudocolored pixels from the areas within the plasma membrane in which both TrkA and the corresponding subtype of lipid rafts pixel value exceed the mean. Scale bar represents 5 μm. Graphs show the average values (mean±SEM) obtained for PC and ICQ. Plots in (A) represent n = 18–26 neurons from three independent assays. Plots in (B) show the results obtained for 15 cells from two independent experiments. Plots in (C) represent n = 21–35 neurons from three independent assays. Plots in (D) represent 16–23 neurons from two independent assays. Two-tailed unpaired T-test, * P

    Article Snippet: In brief, 340 μL of rat tail collagen I (BD Biosciences, San Jose, CA) were mixed with 40 μL of 10x MEM, 10 μL of HEPES or the vCKBPs at a final concentration of 50 nM, and mouse NGF 2.5S (N-100, Alomone labs, Jerusalem, Israel) at a final concentration of 0.25 nM.

    Techniques: Immunofluorescence, Confocal Microscopy, Two Tailed Test