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  • 91
    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, 2024-06
    91/100 stars
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    85
    Santa Cruz Biotechnology dpysl3 sirna
    ONCOMINE™ database analysis of <t> DPYSL3 </t> gene expression
    Dpysl3 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dpysl3 sirna/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dpysl3 sirna - by Bioz Stars, 2024-06
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    92
    Santa Cruz Biotechnology crmp 4
    ONCOMINE™ database analysis of <t> DPYSL3 </t> gene expression
    Crmp 4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crmp 4/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crmp 4 - by Bioz Stars, 2024-06
    92/100 stars
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    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, 2024-06
    91/100 stars
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    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 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, 2024-06
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    Image Search Results


    ONCOMINE™ database analysis of  DPYSL3  gene expression

    Journal: Oncotarget

    Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

    doi: 10.18632/oncotarget.8290

    Figure Lengend Snippet: ONCOMINE™ database analysis of DPYSL3 gene expression

    Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

    Techniques:

    A. Public dataset was extracted from Oncomine™ and graphed in to three groups. The numbers in white indicate the median value and patient case number is indicated on x-axel. B. cDNA microarray dataset from a previously published report was re-analyzed. Data represent the Mean from different patient groups, including benign prostate specimens (normal, n = 5), primary cancers (n = 23), tumors after hormone therapy (n = 17), metastasis (n = 9) and castration-resistant tumors (n = 3). The errors bars indicate the standard error of mean (SEM). The asterisk indicates a significant difference compared to other groups (p < 0.05, student's t -test). C. Quantitative analysis of DPYSL3 variants in prostate cancers were conducted using total RNAs extracted from frozen tumor specimens and the individually matched nonmalignant compartments, as described . The expression levels of DPYSL3 variants were normalized against the epithelium-specific gene KRT18 before the relative values were calculated. The relative ratio of gene expression level in malignant compared to benign tissues was presented as fold induction. Error bar represents the SEM. The asterisk indicates a significant difference compared to the ‘normal’ group (p < 0.05, student's t -test). D. Western blot assays were used to evaluate CRMP4 protein expression in 3 pairs of prostate specimens. Cell lysates from 293T cells overexpressing CRMP4a and CRMP4b were used to as positive control. Actin blot served as protein loading control. Data represent two separate experiments.

    Journal: Oncotarget

    Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

    doi: 10.18632/oncotarget.8290

    Figure Lengend Snippet: A. Public dataset was extracted from Oncomine™ and graphed in to three groups. The numbers in white indicate the median value and patient case number is indicated on x-axel. B. cDNA microarray dataset from a previously published report was re-analyzed. Data represent the Mean from different patient groups, including benign prostate specimens (normal, n = 5), primary cancers (n = 23), tumors after hormone therapy (n = 17), metastasis (n = 9) and castration-resistant tumors (n = 3). The errors bars indicate the standard error of mean (SEM). The asterisk indicates a significant difference compared to other groups (p < 0.05, student's t -test). C. Quantitative analysis of DPYSL3 variants in prostate cancers were conducted using total RNAs extracted from frozen tumor specimens and the individually matched nonmalignant compartments, as described . The expression levels of DPYSL3 variants were normalized against the epithelium-specific gene KRT18 before the relative values were calculated. The relative ratio of gene expression level in malignant compared to benign tissues was presented as fold induction. Error bar represents the SEM. The asterisk indicates a significant difference compared to the ‘normal’ group (p < 0.05, student's t -test). D. Western blot assays were used to evaluate CRMP4 protein expression in 3 pairs of prostate specimens. Cell lysates from 293T cells overexpressing CRMP4a and CRMP4b were used to as positive control. Actin blot served as protein loading control. Data represent two separate experiments.

    Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

    Techniques: Microarray, Expressing, Western Blot, Positive Control

     DPYSL3  saRNA target sequence and genomic location on Chromosome 5

    Journal: Oncotarget

    Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

    doi: 10.18632/oncotarget.8290

    Figure Lengend Snippet: DPYSL3 saRNA target sequence and genomic location on Chromosome 5

    Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

    Techniques: Sequencing

     DPYSL3  saRNA suppresses tumor metastasis in vivo

    Journal: Oncotarget

    Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

    doi: 10.18632/oncotarget.8290

    Figure Lengend Snippet: DPYSL3 saRNA suppresses tumor metastasis in vivo

    Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

    Techniques:

    A. Representing H&E section images from major organs as indicated harvested from animals received the APT-saV2-9 or the scramble control. The green arrows indicate metastatic cancer cells in left lung (a), liver (b) and lymph node (c) sections from animals received the control saRNA. No metastatic cancer cells were seen in kidney sections or other organs. B. The whole right lobes of lung harvested from animals treated with the scramble or sa V2-9 conjugates were homogenized and protein extracts were collected for luciferase activity measurement. The readings were normalized with the corresponding protein concentrations expressed as relative LUC reading/mg lung proteins. Data presented are MEAN ± SEM and the asterisk indicates a significant difference compared to the scramble control (P < 0.05, Student's t-test). C. Total RNAs were extracted from xenograft tissues and subjected to qPCR analysis for DPYSl3 gene expression. After normalized with KRT18 gene expression levels, relative fold induction against the value in the samples from the scramble control group was calculated and presented as MEAN ± SEM. The asterisk indicates a significant difference compared to the scramble control (p < 0.05, student's t -test). D. Total proteins were extracted from xenograft tumors and subjected to western blot assays for CRMP4a protein expression. Actin blot served as protein loading control. E. Xenograft tumors obtained from animals treated with the scramble or saV2-9 conjugates were processed for H&E staining, Ki-67 and BrdU immunohistochemical staining. Representing microscopic images were taken from a 200x magnification.

    Journal: Oncotarget

    Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

    doi: 10.18632/oncotarget.8290

    Figure Lengend Snippet: A. Representing H&E section images from major organs as indicated harvested from animals received the APT-saV2-9 or the scramble control. The green arrows indicate metastatic cancer cells in left lung (a), liver (b) and lymph node (c) sections from animals received the control saRNA. No metastatic cancer cells were seen in kidney sections or other organs. B. The whole right lobes of lung harvested from animals treated with the scramble or sa V2-9 conjugates were homogenized and protein extracts were collected for luciferase activity measurement. The readings were normalized with the corresponding protein concentrations expressed as relative LUC reading/mg lung proteins. Data presented are MEAN ± SEM and the asterisk indicates a significant difference compared to the scramble control (P < 0.05, Student's t-test). C. Total RNAs were extracted from xenograft tissues and subjected to qPCR analysis for DPYSl3 gene expression. After normalized with KRT18 gene expression levels, relative fold induction against the value in the samples from the scramble control group was calculated and presented as MEAN ± SEM. The asterisk indicates a significant difference compared to the scramble control (p < 0.05, student's t -test). D. Total proteins were extracted from xenograft tumors and subjected to western blot assays for CRMP4a protein expression. Actin blot served as protein loading control. E. Xenograft tumors obtained from animals treated with the scramble or saV2-9 conjugates were processed for H&E staining, Ki-67 and BrdU immunohistochemical staining. Representing microscopic images were taken from a 200x magnification.

    Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

    Techniques: Luciferase, Activity Assay, Expressing, Western Blot, Staining, Immunohistochemical staining

    ( 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 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.04.11.036863

    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 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.

    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’ elsewhere), 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; 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) ( EFNB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), 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 25252), 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). 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.04.11.036863

    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). 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.

    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’ elsewhere), 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; 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) ( EFNB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), 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 25252), 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