bdnf treatment  (Alomone Labs)


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    Alomone Labs bdnf treatment
    ( A ) Lysates <t>from</t> <t>DCRNs</t> cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Bdnf Treatment, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bdnf treatment/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bdnf treatment - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons"

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064890

    ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Figure Legend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

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

    ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).
    Figure Legend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

    Techniques Used: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

    ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

    Techniques Used: Sandwich ELISA, Sequencing, Cell Culture, Incubation

    ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

    Techniques Used: Cell Culture, Sandwich ELISA

    bdnf  (Alomone Labs)


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    Alomone Labs bdnf
    Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bdnf/product/Alomone Labs
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    86/100 stars

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    bdnf  (Alomone Labs)


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    Alomone Labs bdnf
    Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bdnf/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    rabbit polyclonal anti bdnf  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti bdnf
    Rabbit Polyclonal Anti Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti bdnf/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    86/100 stars

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    bdnf treatment  (Alomone Labs)


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    Structured Review

    Alomone Labs bdnf treatment
    ( A ) Lysates <t>from</t> <t>DCRNs</t> cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Bdnf Treatment, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bdnf treatment/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bdnf treatment - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons"

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064890

    ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Figure Legend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

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

    ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).
    Figure Legend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

    Techniques Used: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

    ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

    Techniques Used: Sandwich ELISA, Sequencing, Cell Culture, Incubation

    ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

    Techniques Used: Cell Culture, Sandwich ELISA

    bdnf  (Alomone Labs)


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    Alomone Labs bdnf
    ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 <t>ng/m</t> <t>NGF</t> and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bdnf/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bdnf - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons"

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064890

    ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Figure Legend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

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

    ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).
    Figure Legend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

    Techniques Used: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

    ( A ) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). ( B ) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.
    Figure Legend Snippet: ( A ) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). ( B ) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.

    Techniques Used: Cell Culture, Transfection

    ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

    Techniques Used: Sandwich ELISA, Sequencing, Cell Culture, Incubation

    ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).
    Figure Legend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

    Techniques Used: Cell Culture, Sandwich ELISA

    E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).
    Figure Legend Snippet: E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).

    Techniques Used: Mutagenesis, Cell Culture

    rabbit anti pro bdnf ab  (Alomone Labs)


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    Alomone Labs rabbit anti pro bdnf ab
    (A): RT-PCR of <t>BDNF,</t> its <t>high</t> <t>(TrkB)</t> and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.
    Rabbit Anti Pro Bdnf Ab, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival"

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025097

    (A): RT-PCR of BDNF, its high (TrkB) and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.
    Figure Legend Snippet: (A): RT-PCR of BDNF, its high (TrkB) and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Expressing

    BDNF release and effect of exogenous BDNF,  pro-BDNF,   TrkB  inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in primary CRC cell lines.
    Figure Legend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in primary CRC cell lines.

    Techniques Used:

    BDNF release and effect of exogenous BDNF,  pro-BDNF,   TrkB  inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.
    Figure Legend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.

    Techniques Used:

    Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.
    Figure Legend Snippet: Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.

    Techniques Used: Confocal Microscopy, Staining, Cell Culture

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
    Figure Legend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Techniques Used: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    recombinant human bdnf  (Alomone Labs)


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    Alomone Labs recombinant human bdnf
    (A) Role of endogenous <t>BDNF</t> and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF <t>(+),</t> <t>K252a</t> (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
    Recombinant Human Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human bdnf/product/Alomone Labs
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    recombinant human bdnf - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival"

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025097

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
    Figure Legend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Techniques Used: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).
    Figure Legend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Techniques Used: Double Staining, Confocal Microscopy, Staining, Recombinant

    recombinant pro bdnf  (Alomone Labs)


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    Alomone Labs recombinant pro bdnf
    (A) Role of endogenous <t>BDNF</t> and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF <t>(+),</t> <t>K252a</t> (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
    Recombinant Pro Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant pro bdnf/product/Alomone Labs
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    1) Product Images from "Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival"

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025097

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
    Figure Legend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Techniques Used: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).
    Figure Legend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Techniques Used: Double Staining, Confocal Microscopy, Staining, Recombinant

    bdnf  (Alomone Labs)


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    Alomone Labs bdnf
    Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor <t>(BDNF),</t> Netrin-1 <t>and</t> <t>Sema-3a</t> in an in vitro turning assay . (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). T i = initial trajectory; T f = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D) . Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: * P < 0.05; **P < 0.005; Mann-Whitney U -test. Error bars indicate standard error of the mean.
    Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Homer regulates calcium signalling in growth cone turning"

    Article Title: Homer regulates calcium signalling in growth cone turning

    Journal: Neural Development

    doi: 10.1186/1749-8104-4-29

    Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay . (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). T i = initial trajectory; T f = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D) . Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: * P < 0.05; **P < 0.005; Mann-Whitney U -test. Error bars indicate standard error of the mean.
    Figure Legend Snippet: Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay . (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). T i = initial trajectory; T f = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D) . Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: * P < 0.05; **P < 0.005; Mann-Whitney U -test. Error bars indicate standard error of the mean.

    Techniques Used: Derivative Assay, In Vitro, MANN-WHITNEY

    Specific anti-sense morpholino knockdown of Homer1 expression causes a reversal in growth cone turning . (A) Control morphant and (B) Homer1 morphant growth cones labelled for Homer1b/c (red) and f-actin (green) after 6 hours in culture. (C) Homer1 knockdown in growth cones was quantified by determining pixel intensity corrected for growth cone area and normalised to control morphant growth cones (100%). Numbers indicate growth cones imaged over three separate dishes. (D) Western blot of B-35 neuroblastoma cell protein extracts loaded with control (C), and Homer1 (H) morpholinos. Partial knockdown was achieved at 12 hours and significant knockdown at 24 hours. (E) Average growth cone turning angles after 30 minutes in gradients of Netrin-1, brain derived neurotrophic factor (BDNF) or Sema-3a after a 4 to 6 hour treatment with control morpholino (open bars) or Homer1 morpholino (shaded bars). (F) Control or Homer1 morpholinos did not have any significant effect on overall axon extension rates (compare Figures 1B and 2E). Significant differences from control values are marked as: *** P < 0.0005; Mann-Whitney U -test. Error bars indicate standard error of the mean. Scale bar for (A, B) is 5 μm.
    Figure Legend Snippet: Specific anti-sense morpholino knockdown of Homer1 expression causes a reversal in growth cone turning . (A) Control morphant and (B) Homer1 morphant growth cones labelled for Homer1b/c (red) and f-actin (green) after 6 hours in culture. (C) Homer1 knockdown in growth cones was quantified by determining pixel intensity corrected for growth cone area and normalised to control morphant growth cones (100%). Numbers indicate growth cones imaged over three separate dishes. (D) Western blot of B-35 neuroblastoma cell protein extracts loaded with control (C), and Homer1 (H) morpholinos. Partial knockdown was achieved at 12 hours and significant knockdown at 24 hours. (E) Average growth cone turning angles after 30 minutes in gradients of Netrin-1, brain derived neurotrophic factor (BDNF) or Sema-3a after a 4 to 6 hour treatment with control morpholino (open bars) or Homer1 morpholino (shaded bars). (F) Control or Homer1 morpholinos did not have any significant effect on overall axon extension rates (compare Figures 1B and 2E). Significant differences from control values are marked as: *** P < 0.0005; Mann-Whitney U -test. Error bars indicate standard error of the mean. Scale bar for (A, B) is 5 μm.

    Techniques Used: Expressing, Western Blot, Derivative Assay, MANN-WHITNEY

    Homer signalling operates in a calcium-dependent manner and regulates the operational state of a calcium-calmodulin dependent protein kinase II-calcineurin phosphatase molecular switch . (A, C) Average growth cone turning angles for control (open bars) and Homer (shaded bars) morphant growth cones in response to brain derived neurotrophic factor (BDNF) and Sema-3a gradients following a 20 minute bath application of (A) cyclosporinA (CsA) or (C) KN93 and KN92. (B, D) Axon extension rates were not significantly different amongst control and Homer1 morphants with the same pharmacological treatments. Significant differences from control values are marked as: ** P < 0.005; *** P < 0.0005; Mann-Whitney U -test. Error bars indicate standard error of the mean.
    Figure Legend Snippet: Homer signalling operates in a calcium-dependent manner and regulates the operational state of a calcium-calmodulin dependent protein kinase II-calcineurin phosphatase molecular switch . (A, C) Average growth cone turning angles for control (open bars) and Homer (shaded bars) morphant growth cones in response to brain derived neurotrophic factor (BDNF) and Sema-3a gradients following a 20 minute bath application of (A) cyclosporinA (CsA) or (C) KN93 and KN92. (B, D) Axon extension rates were not significantly different amongst control and Homer1 morphants with the same pharmacological treatments. Significant differences from control values are marked as: ** P < 0.005; *** P < 0.0005; Mann-Whitney U -test. Error bars indicate standard error of the mean.

    Techniques Used: Derivative Assay, MANN-WHITNEY

    Homer1 knockdown perturbs dorsal root ganglia growth cone calcium dynamics . (A) Control (closed red circles) morphant growth cones (n = 9) exposed to a brain derived neurotrophic factor (BDNF) micro-gradient show significantly more calcium flux than Homer1 (closed green circles) morphant growth cones (n = 16). (B) Bath application of thapsigargin 2 minutes after establishment of a BDNF micro-gradient elicited a robust rise in intracellular calcium in Homer morphants (hom morph) but failed to elicit any rise in calcium in control morphants (ctrl morph). Error bars indicate standard error of the mean.
    Figure Legend Snippet: Homer1 knockdown perturbs dorsal root ganglia growth cone calcium dynamics . (A) Control (closed red circles) morphant growth cones (n = 9) exposed to a brain derived neurotrophic factor (BDNF) micro-gradient show significantly more calcium flux than Homer1 (closed green circles) morphant growth cones (n = 16). (B) Bath application of thapsigargin 2 minutes after establishment of a BDNF micro-gradient elicited a robust rise in intracellular calcium in Homer morphants (hom morph) but failed to elicit any rise in calcium in control morphants (ctrl morph). Error bars indicate standard error of the mean.

    Techniques Used: Derivative Assay

    Spontaneous calcium transients and growth cone turning are sensitive to blockage of store-operated channels . (A) Individual control morphant growth cones exhibited sparse spontaneous calcium transients, occurring at a rate of approximately one transient per three minutes. (B) Homer1 morphant growth cones exhibited significantly greater frequency, at a rate of at least one spontaneous transient per minute. (C) A trace from a single Homer1 morphant growth cone showed a decrease in spontaneous calcium transient frequency in the presence of bath applied SKF-96365. (D) Quantification of spontaneous calcium transient frequencies in Homer1 morphant growth cones. Removing calcium from the media (Ca free) or bath application of La 3+ (La) or SKF-96365 (SKF) reduced spontaneous transient frequencies in Homer1 morphant growth cones to control (ctrl) levels. Bath application of a voltage-gated calcium channel (VGCC) inhibitor cocktail or nifedipine alone had little effect on the frequency of spontaneous calcium transients in Homer1 morphant growth cones. (E) Calcium-dependent brain derived neurotrophic factor (BDNF)-induced turning is mediated through store-operated channels. BDNF attraction was abolished when TRPC channels were inactivated with bath application of SKF-96365 or La 3+ . Inhibition of VGCCs with nifedipine or ω-conotoxin-MVIIC had no effect on control and Homer1 morphant growth cone turning. (F) Inhibition of store-operated channels did not alter axon extension rates. Error bars indicate standard error of the mean. Cocktail = nifedipine, ω-conotoxin-MVIIC plus Ni ++ . The scale bar in (C) applies also to (A, B).
    Figure Legend Snippet: Spontaneous calcium transients and growth cone turning are sensitive to blockage of store-operated channels . (A) Individual control morphant growth cones exhibited sparse spontaneous calcium transients, occurring at a rate of approximately one transient per three minutes. (B) Homer1 morphant growth cones exhibited significantly greater frequency, at a rate of at least one spontaneous transient per minute. (C) A trace from a single Homer1 morphant growth cone showed a decrease in spontaneous calcium transient frequency in the presence of bath applied SKF-96365. (D) Quantification of spontaneous calcium transient frequencies in Homer1 morphant growth cones. Removing calcium from the media (Ca free) or bath application of La 3+ (La) or SKF-96365 (SKF) reduced spontaneous transient frequencies in Homer1 morphant growth cones to control (ctrl) levels. Bath application of a voltage-gated calcium channel (VGCC) inhibitor cocktail or nifedipine alone had little effect on the frequency of spontaneous calcium transients in Homer1 morphant growth cones. (E) Calcium-dependent brain derived neurotrophic factor (BDNF)-induced turning is mediated through store-operated channels. BDNF attraction was abolished when TRPC channels were inactivated with bath application of SKF-96365 or La 3+ . Inhibition of VGCCs with nifedipine or ω-conotoxin-MVIIC had no effect on control and Homer1 morphant growth cone turning. (F) Inhibition of store-operated channels did not alter axon extension rates. Error bars indicate standard error of the mean. Cocktail = nifedipine, ω-conotoxin-MVIIC plus Ni ++ . The scale bar in (C) applies also to (A, B).

    Techniques Used: Derivative Assay, Inhibition

    bdnf  (Alomone Labs)


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    Structured Review

    Alomone Labs bdnf
    Role of Trk and p75 NTR in <t>BDNF's</t> effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with the function-blocking antibody of p75 NTR , REX (50 μ g/mL), and BDNF (250 ng/mL) for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. (d) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in SM and treated <t>with</t> <t>k-252a</t> (200 nM) and BDNF (250 ng/mL) for 48 hrs. (e) Dendritic spine density expressed per 10 μ m of apical dendrite. (f) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.
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    Images

    1) Product Images from "Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology"

    Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

    Journal: Neural Plasticity

    doi: 10.1155/2012/578057

    Role of Trk and p75 NTR in BDNF's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with the function-blocking antibody of p75 NTR , REX (50 μ g/mL), and BDNF (250 ng/mL) for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. (d) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in SM and treated with k-252a (200 nM) and BDNF (250 ng/mL) for 48 hrs. (e) Dendritic spine density expressed per 10 μ m of apical dendrite. (f) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.
    Figure Legend Snippet: Role of Trk and p75 NTR in BDNF's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with the function-blocking antibody of p75 NTR , REX (50 μ g/mL), and BDNF (250 ng/mL) for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. (d) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in SM and treated with k-252a (200 nM) and BDNF (250 ng/mL) for 48 hrs. (e) Dendritic spine density expressed per 10 μ m of apical dendrite. (f) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.

    Techniques Used: Blocking Assay

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    Alomone Labs bdnf treatment
    ( A ) Lysates <t>from</t> <t>DCRNs</t> cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
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    Alomone Labs bdnf
    ( A ) Lysates <t>from</t> <t>DCRNs</t> cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
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    Alomone Labs rabbit polyclonal anti bdnf
    ( A ) Lysates <t>from</t> <t>DCRNs</t> cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml <t>BDNF</t> were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).
    Rabbit Polyclonal Anti Bdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti pro bdnf ab
    (A): RT-PCR of <t>BDNF,</t> its <t>high</t> <t>(TrkB)</t> and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.
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    Alomone Labs recombinant human bdnf
    (A) Role of endogenous <t>BDNF</t> and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF <t>(+),</t> <t>K252a</t> (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
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    Alomone Labs recombinant pro bdnf
    (A) Role of endogenous <t>BDNF</t> and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF <t>(+),</t> <t>K252a</t> (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.
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    ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

    Journal: PLoS ONE

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    doi: 10.1371/journal.pone.0064890

    Figure Lengend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

    Article Snippet: K252a (200 nM) (Alomone Labs) and MK-1775 (300 nM) (Axon MedChem), both prepared in dimethyl sulfoxide, were added to the culture medium 10 min before BDNF treatment (DCRNs).

    Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

    ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

    Journal: PLoS ONE

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    doi: 10.1371/journal.pone.0064890

    Figure Lengend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

    Article Snippet: K252a (200 nM) (Alomone Labs) and MK-1775 (300 nM) (Axon MedChem), both prepared in dimethyl sulfoxide, were added to the culture medium 10 min before BDNF treatment (DCRNs).

    Techniques: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

    ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

    Journal: PLoS ONE

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    doi: 10.1371/journal.pone.0064890

    Figure Lengend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

    Article Snippet: K252a (200 nM) (Alomone Labs) and MK-1775 (300 nM) (Axon MedChem), both prepared in dimethyl sulfoxide, were added to the culture medium 10 min before BDNF treatment (DCRNs).

    Techniques: Sandwich ELISA, Sequencing, Cell Culture, Incubation

    ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

    Journal: PLoS ONE

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

    doi: 10.1371/journal.pone.0064890

    Figure Lengend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

    Article Snippet: K252a (200 nM) (Alomone Labs) and MK-1775 (300 nM) (Axon MedChem), both prepared in dimethyl sulfoxide, were added to the culture medium 10 min before BDNF treatment (DCRNs).

    Techniques: Cell Culture, Sandwich ELISA

    (A): RT-PCR of BDNF, its high (TrkB) and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A): RT-PCR of BDNF, its high (TrkB) and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.

    Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Expressing

    BDNF release and effect of exogenous BDNF,  pro-BDNF,   TrkB  inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in primary CRC cell lines.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in primary CRC cell lines.

    Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

    Techniques:

    BDNF release and effect of exogenous BDNF,  pro-BDNF,   TrkB  inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.

    Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

    Techniques:

    Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.

    Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

    Techniques: Confocal Microscopy, Staining, Cell Culture

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

    Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

    Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

    Techniques: Double Staining, Confocal Microscopy, Staining, Recombinant

    (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

    Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

    Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Journal: PLoS ONE

    Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

    doi: 10.1371/journal.pone.0025097

    Figure Lengend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

    Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

    Techniques: Double Staining, Confocal Microscopy, Staining, Recombinant