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    Alomone Labs ngf
    Neurite outgrowth induced by treatment with Y27632 (a) and <t>nerve</t> <t>growth</t> <t>factor</t> <t>(NGF)</t> (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.
    Ngf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) 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

    2) 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

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    Alomone Labs nerve growth factor ngf
    <t>BDNF-induced</t> NR2C upregulation by TrkB activation. Cells were cultured in low KCl and treated with or without BDNF (100 ng/ml in A and 50 ng/ml in B-D and F ) for 96 h. A , Levels of indicated mRNAs were quantitated by RNA blotting ( n = 4). B , Cell lysates (40 μg) were immunoblotted with anti-panNR2 antibody or anti-NR1 antibody. Molecular sizes (kilodaltons) of protein makers are indicated on the left. C , P2 membrane fractions were isolated, solubilized, and immunoprecipitated (IP) with anti-NR1 antibody, followed by immnoblotting with anti-panNR2 antibody. D , Cell-surface proteins were biotinylated with Sulfo-NHS-SS-Biotin. Cell lysates were solubilized, precipitated with NeutrAvidin beads, and immunoblotted with anti-panNR2 antibody. E , Cells were cultured in low KCl and treated with BDNF, <t>NGF,</t> NT-3, or NT-4 (50 ng/ml each) for 96 h. Levels of NR2C mRNA were quantitated ( n = 4). F , Granule cells were prepared from TrkB - / - knock-out mice(KO) and their wild-type (WT) littermates. NR2C and TrkB mRNAs were analyzed by RNA blotting. Ethidium bromide-stained 18s rRNA is also indicated. * p
    Nerve Growth Factor Ngf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs n m bdnf pro domain
    Differential binding of <t>pro-BDNF</t> and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 <t>n</t> m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor
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    BDNF-induced NR2C upregulation by TrkB activation. Cells were cultured in low KCl and treated with or without BDNF (100 ng/ml in A and 50 ng/ml in B-D and F ) for 96 h. A , Levels of indicated mRNAs were quantitated by RNA blotting ( n = 4). B , Cell lysates (40 μg) were immunoblotted with anti-panNR2 antibody or anti-NR1 antibody. Molecular sizes (kilodaltons) of protein makers are indicated on the left. C , P2 membrane fractions were isolated, solubilized, and immunoprecipitated (IP) with anti-NR1 antibody, followed by immnoblotting with anti-panNR2 antibody. D , Cell-surface proteins were biotinylated with Sulfo-NHS-SS-Biotin. Cell lysates were solubilized, precipitated with NeutrAvidin beads, and immunoblotted with anti-panNR2 antibody. E , Cells were cultured in low KCl and treated with BDNF, NGF, NT-3, or NT-4 (50 ng/ml each) for 96 h. Levels of NR2C mRNA were quantitated ( n = 4). F , Granule cells were prepared from TrkB - / - knock-out mice(KO) and their wild-type (WT) littermates. NR2C and TrkB mRNAs were analyzed by RNA blotting. Ethidium bromide-stained 18s rRNA is also indicated. * p

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Depolarization Controls Brain-Derived Neurotrophic Factor-Induced Upregulation of NR2C NMDA Receptor via Calcineurin Signaling

    doi: 10.1523/JNEUROSCI.2191-05.2005

    Figure Lengend Snippet: BDNF-induced NR2C upregulation by TrkB activation. Cells were cultured in low KCl and treated with or without BDNF (100 ng/ml in A and 50 ng/ml in B-D and F ) for 96 h. A , Levels of indicated mRNAs were quantitated by RNA blotting ( n = 4). B , Cell lysates (40 μg) were immunoblotted with anti-panNR2 antibody or anti-NR1 antibody. Molecular sizes (kilodaltons) of protein makers are indicated on the left. C , P2 membrane fractions were isolated, solubilized, and immunoprecipitated (IP) with anti-NR1 antibody, followed by immnoblotting with anti-panNR2 antibody. D , Cell-surface proteins were biotinylated with Sulfo-NHS-SS-Biotin. Cell lysates were solubilized, precipitated with NeutrAvidin beads, and immunoblotted with anti-panNR2 antibody. E , Cells were cultured in low KCl and treated with BDNF, NGF, NT-3, or NT-4 (50 ng/ml each) for 96 h. Levels of NR2C mRNA were quantitated ( n = 4). F , Granule cells were prepared from TrkB - / - knock-out mice(KO) and their wild-type (WT) littermates. NR2C and TrkB mRNAs were analyzed by RNA blotting. Ethidium bromide-stained 18s rRNA is also indicated. * p

    Article Snippet: Slices were treated with or without 1 μ m FK506 for 96 h, and 0.5 vol of the culture medium was changed every 2 d. Reagents and inhibitors were purchased from the following sources: BDNF (Peprotech, London, UK); neurotrophin-3 (NT-3), NT-4, and nerve growth factor (NGF) (Alomone Laboratories, Jerusalem, Israel); neuregulin-β (NeoMarkers, Fremont, CA); nifedipine (Nacalai Tesque, Kyoto, Japan); KN62 (Tocris, Bristol, UK); and U0126, SB203580, FK506, K252a, , PD98059, cyclosporin A, rapamycin, and bisindolylmaleimide I (Calbiochem, San Diego, CA).

    Techniques: Activation Assay, Cell Culture, Isolation, Immunoprecipitation, Knock-Out, Mouse Assay, Staining

    Electrical stimulation based bFGF release and bioactivity assay (A) bFGF release profile during sequential electrical stimulation (200 mV/mm, 1Hz, 5 min) and incubation (5 min) periods (B) Bioactivity of released bFGF complexes compared to recombinant human bFGF, biotinylated bFGF, and control groups with no growth factor based on BALB/c cell proliferation (* p

    Journal: Frontiers in Chemistry

    Article Title: Electrically Stimulated Tunable Drug Delivery From Polypyrrole-Coated Polyvinylidene Fluoride

    doi: 10.3389/fchem.2021.599631

    Figure Lengend Snippet: Electrical stimulation based bFGF release and bioactivity assay (A) bFGF release profile during sequential electrical stimulation (200 mV/mm, 1Hz, 5 min) and incubation (5 min) periods (B) Bioactivity of released bFGF complexes compared to recombinant human bFGF, biotinylated bFGF, and control groups with no growth factor based on BALB/c cell proliferation (* p

    Article Snippet: A similar procedure was used for loading a solution of biotinylated Nerve Growth Factor (NGF, Alomone Labs, Jerusalem, Israel) at a concentration of 120 ng/ml in deionized water into a different group of PPy-coated PVDF fibers. illustrates the final schematic of the chemical structure of drug carrier platform containing streptavidin conjugated to biotinylated growth factor and anchored to the biotin incorporated within the PPy coating on the PVDF fibers.

    Techniques: Incubation, Recombinant

    Schematic representation of electrosensitive drug delivery coating. Illustration of the chemical structure of the PPy coating containing sodium p -toluenesulfate (SPTS) and biotin as the primary and secondary doping agents. Streptavidin, with four potential binding sites, covalently binds to the biotin incorporated in the PPy-coating at one site and up to three biotinylated growth factors at the other sites, to function as the drug-complex carrier.

    Journal: Frontiers in Chemistry

    Article Title: Electrically Stimulated Tunable Drug Delivery From Polypyrrole-Coated Polyvinylidene Fluoride

    doi: 10.3389/fchem.2021.599631

    Figure Lengend Snippet: Schematic representation of electrosensitive drug delivery coating. Illustration of the chemical structure of the PPy coating containing sodium p -toluenesulfate (SPTS) and biotin as the primary and secondary doping agents. Streptavidin, with four potential binding sites, covalently binds to the biotin incorporated in the PPy-coating at one site and up to three biotinylated growth factors at the other sites, to function as the drug-complex carrier.

    Article Snippet: A similar procedure was used for loading a solution of biotinylated Nerve Growth Factor (NGF, Alomone Labs, Jerusalem, Israel) at a concentration of 120 ng/ml in deionized water into a different group of PPy-coated PVDF fibers. illustrates the final schematic of the chemical structure of drug carrier platform containing streptavidin conjugated to biotinylated growth factor and anchored to the biotin incorporated within the PPy coating on the PVDF fibers.

    Techniques: Binding Assay

    Electrical stimulation based NGF release and bioactivity assay (A) Streptavidin tethered biotinylated NGF release from PPy coated PVDF fibers during sequential electrical stimulation at 200 mV/mm, for 5 min and 5 min of incubation without stimulation (B) Bioactivity of released NGF compared to recombinant human NGF, biotinylated NGF, and control groups based on PC12 cell proliferation (*, p

    Journal: Frontiers in Chemistry

    Article Title: Electrically Stimulated Tunable Drug Delivery From Polypyrrole-Coated Polyvinylidene Fluoride

    doi: 10.3389/fchem.2021.599631

    Figure Lengend Snippet: Electrical stimulation based NGF release and bioactivity assay (A) Streptavidin tethered biotinylated NGF release from PPy coated PVDF fibers during sequential electrical stimulation at 200 mV/mm, for 5 min and 5 min of incubation without stimulation (B) Bioactivity of released NGF compared to recombinant human NGF, biotinylated NGF, and control groups based on PC12 cell proliferation (*, p

    Article Snippet: A similar procedure was used for loading a solution of biotinylated Nerve Growth Factor (NGF, Alomone Labs, Jerusalem, Israel) at a concentration of 120 ng/ml in deionized water into a different group of PPy-coated PVDF fibers. illustrates the final schematic of the chemical structure of drug carrier platform containing streptavidin conjugated to biotinylated growth factor and anchored to the biotin incorporated within the PPy coating on the PVDF fibers.

    Techniques: Incubation, Recombinant

    Axon directionality on 1400 nm topographical pitch surface or 0 nm pitch flat surface is not altered by diffuse soluble factors. DRG were plated onto either 0 nm pitch control surfaces or 1400 nm pitch surfaces with a basal media enriched with either NGF, GDNF, BDNF or NT-3. Six days after plating, DRG were fixed, stained and imaged using confocal microscopy. The directionality of axons 1500 µm from the DRG body was quantified. DRG cultured on the 1400 nm pitch topography had 85–100% of their axons parallel to the underlying surface topography, independent of the soluble growth factor that was added to the media. The DRG cultured on the 0 nm pitch control substrates had individual axons that ranged from 0–100% parallel to an arbitrary, consistent axis. Each 1400 nm pitch category was significantly different (** = p

    Journal: Biomolecules

    Article Title: Submicron Topographically Patterned 3D Substrates Enhance Directional Axon Outgrowth of Dorsal Root Ganglia Cultured Ex Vivo

    doi: 10.3390/biom12081059

    Figure Lengend Snippet: Axon directionality on 1400 nm topographical pitch surface or 0 nm pitch flat surface is not altered by diffuse soluble factors. DRG were plated onto either 0 nm pitch control surfaces or 1400 nm pitch surfaces with a basal media enriched with either NGF, GDNF, BDNF or NT-3. Six days after plating, DRG were fixed, stained and imaged using confocal microscopy. The directionality of axons 1500 µm from the DRG body was quantified. DRG cultured on the 1400 nm pitch topography had 85–100% of their axons parallel to the underlying surface topography, independent of the soluble growth factor that was added to the media. The DRG cultured on the 0 nm pitch control substrates had individual axons that ranged from 0–100% parallel to an arbitrary, consistent axis. Each 1400 nm pitch category was significantly different (** = p

    Article Snippet: DRGs were maintained in serum-free media containing 5 ng/mL of nerve growth factor (NGF) (Alomone Labs, Ltd., Jerusalem, Israel) and incubated at 37 °C with 5% CO2 for up to 6 days.

    Techniques: Staining, Confocal Microscopy, Cell Culture

    Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Binding Assay, SPR Assay

    Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Derivative Assay, SPR Assay, Binding Assay

    Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Mutagenesis, Binding Assay, SPR Assay, Concentration Assay