human crmp4 tgfp  (OriGene)


Bioz Verified Symbol OriGene is a verified supplier
Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene human crmp4 tgfp
    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and <t>CRMP4-KO</t> (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Human Crmp4 Tgfp, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human crmp4 tgfp/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human crmp4 tgfp - by Bioz Stars, 2024-07
    91/100 stars

    Images

    1) Product Images from "CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway"

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    Journal: eLife

    doi: 10.7554/eLife.70361

    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Figure Legend Snippet: ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.

    Techniques Used: Immunolabeling, Cell Culture, In Vitro

    ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.
    Figure Legend Snippet: ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY

    Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).
    Figure Legend Snippet: Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).

    Techniques Used: Knock-Out, Expressing, Immunolabeling, Staining

    ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.
    Figure Legend Snippet: ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.

    Techniques Used:

    human crmp4 tgfp  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene human crmp4 tgfp
    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and <t>CRMP4-KO</t> (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Human Crmp4 Tgfp, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human crmp4 tgfp/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human crmp4 tgfp - by Bioz Stars, 2024-07
    91/100 stars

    Images

    1) Product Images from "CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway"

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    Journal: eLife

    doi: 10.7554/eLife.70361

    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Figure Legend Snippet: ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.

    Techniques Used: Immunolabeling, Cell Culture, In Vitro

    ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.
    Figure Legend Snippet: ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY

    Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).
    Figure Legend Snippet: Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).

    Techniques Used: Knock-Out, Expressing, Immunolabeling, Staining

    ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.
    Figure Legend Snippet: ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.

    Techniques Used:

    crmp4  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    OriGene crmp4
    Crmp4, supplied by OriGene, 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/crmp4/product/OriGene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crmp4 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    full length mouse crmp4  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene full length mouse crmp4
    a Top, diagram showing the recruitment of CRMP2-5 adaptor complex <t>(CRMP4</t> highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.
    Full Length Mouse Crmp4, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length mouse crmp4/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    full length mouse crmp4 - by Bioz Stars, 2024-07
    91/100 stars

    Images

    1) Product Images from "Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis"

    Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22603-4

    a Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.
    Figure Legend Snippet: a Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.

    Techniques Used: Sequencing, Binding Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot

    a Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n > 100 puncta per condition). Statistical analysis was performed by one-way ANOVA; NS non significant; P values as indicated. b Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. Scale bar, 20 μm (main) and 10 μm (inset). c Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5 μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10 min, or in cells depleted of Bin1 Amphiphysin (Amph + Bin1 DKD). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 150 puncta per condition). Statistical analysis was performed by one-way ANOVA; * P < 0.05, *** P < 0.001. d Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels from three independent biological experiments. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001. e Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 50 puncta per condition). Statistical analysis was performed by one-way ANOVA; ***, P < 0.001 . f Model: multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.
    Figure Legend Snippet: a Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n > 100 puncta per condition). Statistical analysis was performed by one-way ANOVA; NS non significant; P values as indicated. b Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. Scale bar, 20 μm (main) and 10 μm (inset). c Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5 μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10 min, or in cells depleted of Bin1 Amphiphysin (Amph + Bin1 DKD). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 150 puncta per condition). Statistical analysis was performed by one-way ANOVA; * P < 0.05, *** P < 0.001. d Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels from three independent biological experiments. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001. e Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 50 puncta per condition). Statistical analysis was performed by one-way ANOVA; ***, P < 0.001 . f Model: multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.

    Techniques Used: Negative Control, Binding Assay, Activity Assay

    crmp4  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene crmp4
    The ionizing radiation-resistant (IR) cell lines show <t>CRMP4</t> downregulation and increased survival rate. (A) Heatmap from RNA sequencing results. Twenty representative genes are shown. CRMP4 was identified as a downregulated gene. (B) CRMP4 downregulation in IR-resistant cells. Several colon cancer cell lines were lysed and analyzed by western blotting. IR-cells were established by repeating radiation exposure as described in the Materials and methods. (C) Cells were irradiated to the indicated doses of radiation and allowed to grow for 2 weeks, and cells were stained with crystal violet and scored for the colony-forming capacity of irradiated cells. (D) Apoptosis inhibition in IR-resistant cells. Cells were treated with 5 Gy of radiation, and after 48 h, cells were stained with Annexin-V and propidium iodide (PI) followed by flow cytometry analysis. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V + /PI − , Annexin-V + /PI + ) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (E) Reduced G2M phase accumulation in IR-resistant cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.
    Crmp4, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crmp4/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crmp4 - by Bioz Stars, 2024-07
    91/100 stars

    Images

    1) Product Images from "Collapsin response mediator protein 4 enhances the radiosensitivity of colon cancer cells through calcium-mediated cell signaling"

    Article Title: Collapsin response mediator protein 4 enhances the radiosensitivity of colon cancer cells through calcium-mediated cell signaling

    Journal: Oncology Reports

    doi: 10.3892/or.2021.7957

    The ionizing radiation-resistant (IR) cell lines show CRMP4 downregulation and increased survival rate. (A) Heatmap from RNA sequencing results. Twenty representative genes are shown. CRMP4 was identified as a downregulated gene. (B) CRMP4 downregulation in IR-resistant cells. Several colon cancer cell lines were lysed and analyzed by western blotting. IR-cells were established by repeating radiation exposure as described in the Materials and methods. (C) Cells were irradiated to the indicated doses of radiation and allowed to grow for 2 weeks, and cells were stained with crystal violet and scored for the colony-forming capacity of irradiated cells. (D) Apoptosis inhibition in IR-resistant cells. Cells were treated with 5 Gy of radiation, and after 48 h, cells were stained with Annexin-V and propidium iodide (PI) followed by flow cytometry analysis. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V + /PI − , Annexin-V + /PI + ) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (E) Reduced G2M phase accumulation in IR-resistant cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.
    Figure Legend Snippet: The ionizing radiation-resistant (IR) cell lines show CRMP4 downregulation and increased survival rate. (A) Heatmap from RNA sequencing results. Twenty representative genes are shown. CRMP4 was identified as a downregulated gene. (B) CRMP4 downregulation in IR-resistant cells. Several colon cancer cell lines were lysed and analyzed by western blotting. IR-cells were established by repeating radiation exposure as described in the Materials and methods. (C) Cells were irradiated to the indicated doses of radiation and allowed to grow for 2 weeks, and cells were stained with crystal violet and scored for the colony-forming capacity of irradiated cells. (D) Apoptosis inhibition in IR-resistant cells. Cells were treated with 5 Gy of radiation, and after 48 h, cells were stained with Annexin-V and propidium iodide (PI) followed by flow cytometry analysis. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V + /PI − , Annexin-V + /PI + ) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (E) Reduced G2M phase accumulation in IR-resistant cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.

    Techniques Used: RNA Sequencing Assay, Western Blot, Irradiation, Staining, Inhibition, Flow Cytometry

    CRMP4-deficiency enhances resistance to radiation. (A) Increased radioresistance in siCRMP4 cells. After cells were transfected with control siRNA (Mock) or CRMP4 siRNA, cells were treated with radiation (5 Gy) and analyzed by clonogenic assay. Representative images are shown on the left, and a quantitative graph is shown on the right. Student's t-test was performed. *P<0.05, **P<0.01. (B) Inhibition of apoptosis in siCRMP4 cells. Cells were treated with 5 Gy of radiation for 48 h, stained with Annexin-V and propidium iodide (PI), and the percentages of apoptotic cells were measured by flow cytometry. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V + /PI − , Annexin-V + /PI + ) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (C) Reduced G2M phase accumulation in siCRMP4 cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell-cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.
    Figure Legend Snippet: CRMP4-deficiency enhances resistance to radiation. (A) Increased radioresistance in siCRMP4 cells. After cells were transfected with control siRNA (Mock) or CRMP4 siRNA, cells were treated with radiation (5 Gy) and analyzed by clonogenic assay. Representative images are shown on the left, and a quantitative graph is shown on the right. Student's t-test was performed. *P<0.05, **P<0.01. (B) Inhibition of apoptosis in siCRMP4 cells. Cells were treated with 5 Gy of radiation for 48 h, stained with Annexin-V and propidium iodide (PI), and the percentages of apoptotic cells were measured by flow cytometry. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V + /PI − , Annexin-V + /PI + ) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (C) Reduced G2M phase accumulation in siCRMP4 cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell-cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.

    Techniques Used: Transfection, Clonogenic Assay, Inhibition, Staining, Flow Cytometry

    CRMP4-deficiency inhibits mitochondrial membrane depolarization under radiation exposure. (A) Inhibition of cytochrome c (CytC) release and PARP cleavage (C-PARP) in IR-resistant cells. After cells were exposed to 5 Gy of radiation for 0–72 h, cytosolic fractions were isolated and western blotting was conducted. (B) Inhibition of cytochrome c release and PARP cleavage in shCRMP4 cells. After cells were exposed to 5 Gy of radiation, cytosolic fractions were isolated and western blotting was conducted. (C) MMP depolarization was significantly increased in parental cells but not in ionizing radiation (IR)-resistant cells. After cells were exposed to 5 Gy of radiation, cells were stained with DIOC6(3) (3,3′-dihexyloxacarbocyanine iodide) fluorescent dye and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. (D) After cells were exposed to 5 Gy of radiation for 72 h, cells were analyzed by flow cytometry using DiOC6(3). MMP depolarization was strongly increased in shControl cells, but it was weakly increased in shCRMP4 cells. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase.
    Figure Legend Snippet: CRMP4-deficiency inhibits mitochondrial membrane depolarization under radiation exposure. (A) Inhibition of cytochrome c (CytC) release and PARP cleavage (C-PARP) in IR-resistant cells. After cells were exposed to 5 Gy of radiation for 0–72 h, cytosolic fractions were isolated and western blotting was conducted. (B) Inhibition of cytochrome c release and PARP cleavage in shCRMP4 cells. After cells were exposed to 5 Gy of radiation, cytosolic fractions were isolated and western blotting was conducted. (C) MMP depolarization was significantly increased in parental cells but not in ionizing radiation (IR)-resistant cells. After cells were exposed to 5 Gy of radiation, cells were stained with DIOC6(3) (3,3′-dihexyloxacarbocyanine iodide) fluorescent dye and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. (D) After cells were exposed to 5 Gy of radiation for 72 h, cells were analyzed by flow cytometry using DiOC6(3). MMP depolarization was strongly increased in shControl cells, but it was weakly increased in shCRMP4 cells. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase.

    Techniques Used: Inhibition, Isolation, Western Blot, Staining, Flow Cytometry

    CRMP4-deficiency inhibits the Ca 2+ -mediated cell death pathway. (A) Intracellular Ca 2+ level was not affected by CRMP4-deficiency. Cells were irradiated with 5 Gy of radiation for 72 h, treated with Fluo-3 AM for Ca 2+ detection, and analyzed by flow cytometry. Histograms of intracellular Ca 2+ content are shown on the right. N.S , not significant. (B) Increased cell survival by cyclosporine A (CsA) in radiation-treated cells. Cells were treated with or without CsA (5 µM) for 1 h and exposed to 5 Gy of radiation, and after 14 days, cells were stained with 0.1% crystal violet. (C) CRMP4-deficiency inhibited A23187-mediated cell death. Several cells were treated with the Ca 2+ ionophore A23187 for 24 h, and their proliferation was measured by a plate reader using the WST-1 reagent. The survival rate is expressed as the % of control cells. Student's t-test was performed. *P<0.05, **P<0.01. (D) Cytochrome c (CytC) release inhibition in CRMP4-downregulated cells. After cells were treated with A23187 (0–2 µM) for 12 h, the cytosolic fractions were isolated and western blotting was conducted. CRMP4-deficient ionizing radiation (IR)-resistant and shCRMP4 cells showed a decreased cytochrome c release. (E) MMP depolarization was increased in shControl cells but not in shCRMP4 cells. After cells were treated with A23187 for 24 h, cells were stained with DIOC6(3) and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.
    Figure Legend Snippet: CRMP4-deficiency inhibits the Ca 2+ -mediated cell death pathway. (A) Intracellular Ca 2+ level was not affected by CRMP4-deficiency. Cells were irradiated with 5 Gy of radiation for 72 h, treated with Fluo-3 AM for Ca 2+ detection, and analyzed by flow cytometry. Histograms of intracellular Ca 2+ content are shown on the right. N.S , not significant. (B) Increased cell survival by cyclosporine A (CsA) in radiation-treated cells. Cells were treated with or without CsA (5 µM) for 1 h and exposed to 5 Gy of radiation, and after 14 days, cells were stained with 0.1% crystal violet. (C) CRMP4-deficiency inhibited A23187-mediated cell death. Several cells were treated with the Ca 2+ ionophore A23187 for 24 h, and their proliferation was measured by a plate reader using the WST-1 reagent. The survival rate is expressed as the % of control cells. Student's t-test was performed. *P<0.05, **P<0.01. (D) Cytochrome c (CytC) release inhibition in CRMP4-downregulated cells. After cells were treated with A23187 (0–2 µM) for 12 h, the cytosolic fractions were isolated and western blotting was conducted. CRMP4-deficient ionizing radiation (IR)-resistant and shCRMP4 cells showed a decreased cytochrome c release. (E) MMP depolarization was increased in shControl cells but not in shCRMP4 cells. After cells were treated with A23187 for 24 h, cells were stained with DIOC6(3) and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.

    Techniques Used: Irradiation, Flow Cytometry, Staining, Inhibition, Isolation, Western Blot

    The Ca 2+ chelator, BAPTA-AM, downregulates CRMP4 and inhibits MMP depolarization. (A) CRMP4 downregulation by BAPTA-AM treatment. SW620 cells were treated with BAPTA-AM (0–20 µM) for 24 h, and western blotting was conducted. (B) A23187-mediated cytochrome c (CytC) release was inhibited by BAPTA-AM. SW620 cells were treated with A23187 (+, 1 µM; ++, 2 µM) in the absence or presence of BAPTA-AM (5 µM) for 12 h, and mitochondrial and cytosolic fractions were isolated for western blotting. (C) Radiation-mediated PARP cleavage (C-PARP) was blocked by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cell lysates were analyzed by western blotting. (D) Radiation-mediated MMP depolarization was inhibited by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cells were stained with DiOC6(3) for flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.
    Figure Legend Snippet: The Ca 2+ chelator, BAPTA-AM, downregulates CRMP4 and inhibits MMP depolarization. (A) CRMP4 downregulation by BAPTA-AM treatment. SW620 cells were treated with BAPTA-AM (0–20 µM) for 24 h, and western blotting was conducted. (B) A23187-mediated cytochrome c (CytC) release was inhibited by BAPTA-AM. SW620 cells were treated with A23187 (+, 1 µM; ++, 2 µM) in the absence or presence of BAPTA-AM (5 µM) for 12 h, and mitochondrial and cytosolic fractions were isolated for western blotting. (C) Radiation-mediated PARP cleavage (C-PARP) was blocked by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cell lysates were analyzed by western blotting. (D) Radiation-mediated MMP depolarization was inhibited by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cells were stained with DiOC6(3) for flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.

    Techniques Used: Western Blot, Isolation, Staining, Flow Cytometry

    High-dose BAPTA-AM enhances cell death in CRMP4-deficient cells. (A) Cells were treated with the indicated doses of BAPTA-AM for 72 h, and cells were stained with crystal violet. Representative images are shown. (B) Cell survival inhibition in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM (>10 µM) for 48 h, cells were incubated with the WST-1 reagent, and their absorbances at OD 450 were read using a plate reader. One-way ANOVA with Tukey's post hoc test between control, ionizing radiation (IR)-resistant, and shCRMP4 groups were performed. *P<0.05, **P<0.01. (C) Cleaved PARP (C-PARP) induction in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM for 48 h, cell lysates were analyzed by western blotting. C-PARP was strongly increased in IR-resistant and shCRMP4 cells compared to control cells. CRMP4, collapsin response mediator protein 4; PARP, polyADP-ribose polymerase.
    Figure Legend Snippet: High-dose BAPTA-AM enhances cell death in CRMP4-deficient cells. (A) Cells were treated with the indicated doses of BAPTA-AM for 72 h, and cells were stained with crystal violet. Representative images are shown. (B) Cell survival inhibition in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM (>10 µM) for 48 h, cells were incubated with the WST-1 reagent, and their absorbances at OD 450 were read using a plate reader. One-way ANOVA with Tukey's post hoc test between control, ionizing radiation (IR)-resistant, and shCRMP4 groups were performed. *P<0.05, **P<0.01. (C) Cleaved PARP (C-PARP) induction in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM for 48 h, cell lysates were analyzed by western blotting. C-PARP was strongly increased in IR-resistant and shCRMP4 cells compared to control cells. CRMP4, collapsin response mediator protein 4; PARP, polyADP-ribose polymerase.

    Techniques Used: Staining, Inhibition, Incubation, Western Blot

    Radiation-mediated CRMP4 downregulation inhibits the mitochondrial membrane depolarization followed by apoptosis induction. Radiation- or Ca 2+ ionophore A23187-mediated improper intracellular Ca 2+ influx collapses the cellular Ca 2+ -mediated pathway followed by MMP depolarization. Apoptosis resulting from radiation or A23187 treatment is diminished by CRMP4-deficiency and low levels of the intracellular Ca 2+ chelator, BAPTA-AM (<5 µM). Conversly, significant cell death can be triggered by CRMP4-deficiency and a high dose of BAPTA-AM (>10 µM). This suggests that CRMP4 plays an important role in the Ca 2+ -mediated cell death pathway. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase; IR, ionizing radiation.
    Figure Legend Snippet: Radiation-mediated CRMP4 downregulation inhibits the mitochondrial membrane depolarization followed by apoptosis induction. Radiation- or Ca 2+ ionophore A23187-mediated improper intracellular Ca 2+ influx collapses the cellular Ca 2+ -mediated pathway followed by MMP depolarization. Apoptosis resulting from radiation or A23187 treatment is diminished by CRMP4-deficiency and low levels of the intracellular Ca 2+ chelator, BAPTA-AM (<5 µM). Conversly, significant cell death can be triggered by CRMP4-deficiency and a high dose of BAPTA-AM (>10 µM). This suggests that CRMP4 plays an important role in the Ca 2+ -mediated cell death pathway. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase; IR, ionizing radiation.

    Techniques Used:

    crmp4  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene crmp4
    Crmp4, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crmp4/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crmp4 - by Bioz Stars, 2024-07
    91/100 stars

    Images

    hairpin rna shrna  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene hairpin rna shrna
    Hairpin Rna Shrna, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hairpin rna shrna/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hairpin rna shrna - by Bioz Stars, 2024-07
    91/100 stars

    Images

    full length mouse crmp4  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
    Bioz Manufacturer Symbol OriGene manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    OriGene full length mouse crmp4
    a , Top, diagram showing the recruitment of CRMP2-5 adaptor complex <t>(CRMP4</t> highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508-530). Amino acids phosphorylated by GSK3β (T509, T514 and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b , co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c , co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d , Pull-down using GST-SH3 domains of Endophilin A1, A2 or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. ‘input’ lanes correspond to 5% of the cell extracts. e , Recruitment of EGFP-tagged CRMP4 WT, S522D, S522A or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). f , Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =45 cells per condition). g , Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2 and A3 (‘Endophilin TKD), Cdk5 and GSK3α and b (‘CDK5+GSK3α/β TKD’), CRMP4 (‘CRMP4 KD’) or AP2 (‘AP2 KD’) or pre-treated with Cdk5i and/or GSK3i for 5min. Cells were stimulated by 20nM Semaphorin 3A (Sema3A) for 5min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA; NS , non significant; *, P <0.05, ***, P <0.001. Scale bars, 5μm.
    Full Length Mouse Crmp4, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length mouse crmp4/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    full length mouse crmp4 - by Bioz Stars, 2024-07
    91/100 stars

    Images

    1) Product Images from "Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis"

    Article Title: Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

    Journal: bioRxiv

    doi: 10.1101/2020.04.11.036863

    a , Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508-530). Amino acids phosphorylated by GSK3β (T509, T514 and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b , co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c , co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d , Pull-down using GST-SH3 domains of Endophilin A1, A2 or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. ‘input’ lanes correspond to 5% of the cell extracts. e , Recruitment of EGFP-tagged CRMP4 WT, S522D, S522A or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). f , Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =45 cells per condition). g , Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2 and A3 (‘Endophilin TKD), Cdk5 and GSK3α and b (‘CDK5+GSK3α/β TKD’), CRMP4 (‘CRMP4 KD’) or AP2 (‘AP2 KD’) or pre-treated with Cdk5i and/or GSK3i for 5min. Cells were stimulated by 20nM Semaphorin 3A (Sema3A) for 5min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA; NS , non significant; *, P <0.05, ***, P <0.001. Scale bars, 5μm.
    Figure Legend Snippet: a , Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508-530). Amino acids phosphorylated by GSK3β (T509, T514 and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b , co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c , co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d , Pull-down using GST-SH3 domains of Endophilin A1, A2 or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. ‘input’ lanes correspond to 5% of the cell extracts. e , Recruitment of EGFP-tagged CRMP4 WT, S522D, S522A or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). f , Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =45 cells per condition). g , Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2 and A3 (‘Endophilin TKD), Cdk5 and GSK3α and b (‘CDK5+GSK3α/β TKD’), CRMP4 (‘CRMP4 KD’) or AP2 (‘AP2 KD’) or pre-treated with Cdk5i and/or GSK3i for 5min. Cells were stimulated by 20nM Semaphorin 3A (Sema3A) for 5min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA; NS , non significant; *, P <0.05, ***, P <0.001. Scale bars, 5μm.

    Techniques Used: Sequencing, Binding Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot

    a , Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n >100 puncta per condition). b , Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. c , Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10min, or in cells depleted of Bin1 Amphiphysin (‘Amph+Bin1 DKD’). Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from three independent biological experiments ( n >150 puncta per condition). d , Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels. e , Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Right, histograms show the mean ± SEM from three independent biological experiments ( n >50 puncta per condition). f , Model: Multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.
    Figure Legend Snippet: a , Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n >100 puncta per condition). b , Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. c , Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10min, or in cells depleted of Bin1 Amphiphysin (‘Amph+Bin1 DKD’). Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from three independent biological experiments ( n >150 puncta per condition). d , Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels. e , Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Right, histograms show the mean ± SEM from three independent biological experiments ( n >50 puncta per condition). f , Model: Multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.

    Techniques Used: Negative Control, Binding Assay, Activity Assay

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    OriGene human crmp4 tgfp
    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and <t>CRMP4-KO</t> (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Human Crmp4 Tgfp, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human crmp4 tgfp/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human crmp4 tgfp - by Bioz Stars, 2024-07
    91/100 stars
      Buy from Supplier

    86
    OriGene crmp4
    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and <t>CRMP4-KO</t> (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.
    Crmp4, supplied by OriGene, 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/crmp4/product/OriGene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crmp4 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    91
    OriGene full length mouse crmp4
    a Top, diagram showing the recruitment of CRMP2-5 adaptor complex <t>(CRMP4</t> highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.
    Full Length Mouse Crmp4, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length mouse crmp4/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    full length mouse crmp4 - by Bioz Stars, 2024-07
    91/100 stars
      Buy from Supplier

    91
    OriGene hairpin rna shrna
    a Top, diagram showing the recruitment of CRMP2-5 adaptor complex <t>(CRMP4</t> highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.
    Hairpin Rna Shrna, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hairpin rna shrna/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hairpin rna shrna - by Bioz Stars, 2024-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.

    Journal: eLife

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    doi: 10.7554/eLife.70361

    Figure Lengend Snippet: ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.

    Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

    Techniques: Immunolabeling, Cell Culture, In Vitro

    ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.

    Journal: eLife

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    doi: 10.7554/eLife.70361

    Figure Lengend Snippet: ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.

    Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

    Techniques: Knock-Out, Staining, MANN-WHITNEY

    Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).

    Journal: eLife

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    doi: 10.7554/eLife.70361

    Figure Lengend Snippet: Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).

    Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

    Techniques: Knock-Out, Expressing, Immunolabeling, Staining

    ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.

    Journal: eLife

    Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

    doi: 10.7554/eLife.70361

    Figure Lengend Snippet: ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.

    Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

    Techniques:

    a Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.

    Journal: Nature Communications

    Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

    doi: 10.1038/s41467-021-22603-4

    Figure Lengend Snippet: a Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.

    Article Snippet: Full length and truncated genes (all human , unless specified) were amplified and cloned into pDONR201 (Invitrogen) and transferred into pEGFP, pTagRFP-T (called RFP in this study), pMyc, or pGEX-6P2 vectors converted into the Gateway system (pDEST vectors made from a pCI backbone), as appropriate: Endophilin-A2 ( SH3GL1 , IMAGE 3458016) full length and SH3 domain (aa 311-end); Endophilin-A1 ( SH3GL2 iso1 , FLJ 92732) full length and SH3 domain (aa 295-end); Endophilin-A3 ( SH3GL3 iso 1, IMAGE 5197246) full length and SH3 domain (aa 291-end); Bin1, also known as Amphiphysin-II ( BIN1 iso9 , cloned from human brain cDNA library) full length and SH3 domain (aa 366-end); full length CRMP2 ( DPYSL2 , DNASU HsCD00513405), full length CRMP3 ( DPYSL4 , NM_006426 Origene), full length mouse CRMP4 ( DPYSL3 , Origene 1197294), full length CRMP5 ( DPYSL5 , amplified from human brain cDNA library, Novagen), Ephrin receptor A1 cytoplasmic tail (aa 568-976) ( EPHA1 , DNASU HsCD00516390), Ephrin receptor A6 cytoplasmic tail (aa 572-1036) ( EPHA6 , DNASU HsCD00350501), Ephrin receptor B1 cytoplasmic tail (aa 259-346) ( EPHB1 , DNASU HsCD00038738), Ephrin receptor B4 cytoplasmic tail (aa 561-987) ( EPHB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), Semaphorin 4D cytoplasmic tail (aa 756–862) ( SEMA4D , amplified from human brain cDNA library, Novagen), Semaphorin 4F cytoplasmic tail (aa 681–770) ( SEMA4F , DNASU HsCD00041427), Semaphorin 6A cytoplasmic tail (aa 671–1030) ( SEMA6A , Sino Biologica HG11189-M), Semaphorin 6B cytoplasmic tail (aa 616–1021) ( SEMA6B , amplified from human brain cDNA library, Novagen); Semaphorin 6D cytoplasmic tail (aa 684–1073) ( SEMA6D , DNASU HsCD00516397); Plexin B1 cytoplasmic tail (aa 1512–2135) ( PLXNB1 , Addgene 25352), mouse Roundabout homolog 1 cytoplasmic tail (aa 880–1612) ( ROBO1 , DNASU HsCD00295416); Roundabout homolog 3 cytoplasmic tail (aa 912–1386) ( ROBO3 , DNASU HsCD00302878) and Netrin receptor UNC5B cytoplasmic tail (aa 398–945) ( UNC5B , DNASU HsCD294959), EGFP-p27 (Addgene #15192); EGFP-p150Glued (Addgene #36154).

    Techniques: Sequencing, Binding Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot

    a Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n > 100 puncta per condition). Statistical analysis was performed by one-way ANOVA; NS non significant; P values as indicated. b Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. Scale bar, 20 μm (main) and 10 μm (inset). c Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5 μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10 min, or in cells depleted of Bin1 Amphiphysin (Amph + Bin1 DKD). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 150 puncta per condition). Statistical analysis was performed by one-way ANOVA; * P < 0.05, *** P < 0.001. d Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels from three independent biological experiments. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001. e Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 50 puncta per condition). Statistical analysis was performed by one-way ANOVA; ***, P < 0.001 . f Model: multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.

    Journal: Nature Communications

    Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

    doi: 10.1038/s41467-021-22603-4

    Figure Lengend Snippet: a Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n > 100 puncta per condition). Statistical analysis was performed by one-way ANOVA; NS non significant; P values as indicated. b Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. Scale bar, 20 μm (main) and 10 μm (inset). c Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5 μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10 min, or in cells depleted of Bin1 Amphiphysin (Amph + Bin1 DKD). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 150 puncta per condition). Statistical analysis was performed by one-way ANOVA; * P < 0.05, *** P < 0.001. d Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels from three independent biological experiments. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001. e Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 50 puncta per condition). Statistical analysis was performed by one-way ANOVA; ***, P < 0.001 . f Model: multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.

    Article Snippet: Full length and truncated genes (all human , unless specified) were amplified and cloned into pDONR201 (Invitrogen) and transferred into pEGFP, pTagRFP-T (called RFP in this study), pMyc, or pGEX-6P2 vectors converted into the Gateway system (pDEST vectors made from a pCI backbone), as appropriate: Endophilin-A2 ( SH3GL1 , IMAGE 3458016) full length and SH3 domain (aa 311-end); Endophilin-A1 ( SH3GL2 iso1 , FLJ 92732) full length and SH3 domain (aa 295-end); Endophilin-A3 ( SH3GL3 iso 1, IMAGE 5197246) full length and SH3 domain (aa 291-end); Bin1, also known as Amphiphysin-II ( BIN1 iso9 , cloned from human brain cDNA library) full length and SH3 domain (aa 366-end); full length CRMP2 ( DPYSL2 , DNASU HsCD00513405), full length CRMP3 ( DPYSL4 , NM_006426 Origene), full length mouse CRMP4 ( DPYSL3 , Origene 1197294), full length CRMP5 ( DPYSL5 , amplified from human brain cDNA library, Novagen), Ephrin receptor A1 cytoplasmic tail (aa 568-976) ( EPHA1 , DNASU HsCD00516390), Ephrin receptor A6 cytoplasmic tail (aa 572-1036) ( EPHA6 , DNASU HsCD00350501), Ephrin receptor B1 cytoplasmic tail (aa 259-346) ( EPHB1 , DNASU HsCD00038738), Ephrin receptor B4 cytoplasmic tail (aa 561-987) ( EPHB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), Semaphorin 4D cytoplasmic tail (aa 756–862) ( SEMA4D , amplified from human brain cDNA library, Novagen), Semaphorin 4F cytoplasmic tail (aa 681–770) ( SEMA4F , DNASU HsCD00041427), Semaphorin 6A cytoplasmic tail (aa 671–1030) ( SEMA6A , Sino Biologica HG11189-M), Semaphorin 6B cytoplasmic tail (aa 616–1021) ( SEMA6B , amplified from human brain cDNA library, Novagen); Semaphorin 6D cytoplasmic tail (aa 684–1073) ( SEMA6D , DNASU HsCD00516397); Plexin B1 cytoplasmic tail (aa 1512–2135) ( PLXNB1 , Addgene 25352), mouse Roundabout homolog 1 cytoplasmic tail (aa 880–1612) ( ROBO1 , DNASU HsCD00295416); Roundabout homolog 3 cytoplasmic tail (aa 912–1386) ( ROBO3 , DNASU HsCD00302878) and Netrin receptor UNC5B cytoplasmic tail (aa 398–945) ( UNC5B , DNASU HsCD294959), EGFP-p27 (Addgene #15192); EGFP-p150Glued (Addgene #36154).

    Techniques: Negative Control, Binding Assay, Activity Assay