d8e4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc d8e4

    D8e4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug"

    Article Title: Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.109957


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    Techniques Used: Recombinant, Transfection, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Activity Assay, Plasmid Preparation, shRNA, Sequencing, Software

    rabbit monoclonal fgfr1 antibody d8e4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal fgfr1 antibody d8e4

    Rabbit Monoclonal Fgfr1 Antibody D8e4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal fgfr1 antibody d8e4/product/Cell Signaling Technology Inc
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    1) Product Images from "Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs"

    Article Title: Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020107182


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    Techniques Used: Recombinant, Transfection, Molecular Weight, Marker, Protease Inhibitor, Cell Culture, Spectroscopy, Imaging, Plasmid Preparation, Negative Control, Mutagenesis, Clone Assay, Generated, Software, Microscopy

    fgf receptor 1 d8e4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fgf receptor 1 d8e4
    Fgf Receptor 1 D8e4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fgf receptor 1 d8e4 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fgf receptor 1 d8e4 xp rabbit mab
    Fgf Receptor 1 D8e4 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fgf receptor 1 d8e4 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fgf receptor 1 d8e4 xp rabbit mab
    Fgf Receptor 1 D8e4 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fgfr1 d8e4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc fgfr1 d8e4
    KEY RESOURCES TABLE
    Fgfr1 D8e4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Small-molecule and CRISPR screening converge to reveal RTK dependencies in pediatric rhabdoid tumors"

    Article Title: Small-molecule and CRISPR screening converge to reveal RTK dependencies in pediatric rhabdoid tumors

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.07.021

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Cell Viability Assay, Protease Inhibitor, Injection, Sample Prep, Expressing, Activity Assay, Plasmid Preparation, Software, Methylation

    resource source identifier antibodies rabbit monoclonal anti fgf receptor 1 d8e4 cst  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc resource source identifier antibodies rabbit monoclonal anti fgf receptor 1 d8e4 cst
    Resource Source Identifier Antibodies Rabbit Monoclonal Anti Fgf Receptor 1 D8e4 Cst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    d8e4 9740  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc d8e4 9740

    D8e4 9740, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation"

    Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

    Journal: eLife

    doi: 10.7554/eLife.52027


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    Techniques Used: Transfection, Construct, Clone Assay, Plasmid Preparation, Electroporation, shRNA, Recombinant, Sequencing, Immunoprecipitation, Software

    fgf receptor 1 d8e4 xp rabbit mab  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc fgf receptor 1 d8e4 xp rabbit mab
    KEY RESOURCES TABLE
    Fgf Receptor 1 D8e4 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling"

    Article Title: Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.02.014

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Subcloning, Recombinant, Chromatin Immunoprecipitation, Ab Array, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Bicinchoninic Acid Protein Assay, Stable Transfection, Expressing, shRNA, Sequencing, Software

    anti fgfr1 d8e4 xp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti fgfr1 d8e4 xp
    ( a,b ) shRNA against <t>FGFR1,</t> 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.
    Anti Fgfr1 D8e4 Xp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration"

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    Journal: eLife

    doi: 10.7554/eLife.47673

    ( a,b ) shRNA against FGFR1, 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.
    Figure Legend Snippet: ( a,b ) shRNA against FGFR1, 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.

    Techniques Used: shRNA, Dominant Negative Mutation, Negative Control, In Utero, Labeling, Migration, In Vivo

    ( a ) HEK293T cells were transfected with pCAG-FGFR1-GFP, FGFR2-GFP, FGFR3-GFP together with expression vectors for shRNAs against the corresponding receptor or a negative control shRNA (shCtrl). Protein levels were determined by Western blot two days later. ( b ) E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP, FGFR2-GFP or FGFR3-GFP and an expression vectors for shRNA against the corresponding receptor or negative control shRNA. Two days later, CherryFP and FGFRs-GFP were detected by epifluorescence.
    Figure Legend Snippet: ( a ) HEK293T cells were transfected with pCAG-FGFR1-GFP, FGFR2-GFP, FGFR3-GFP together with expression vectors for shRNAs against the corresponding receptor or a negative control shRNA (shCtrl). Protein levels were determined by Western blot two days later. ( b ) E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP, FGFR2-GFP or FGFR3-GFP and an expression vectors for shRNA against the corresponding receptor or negative control shRNA. Two days later, CherryFP and FGFRs-GFP were detected by epifluorescence.

    Techniques Used: Transfection, Expressing, Negative Control, shRNA, Western Blot, In Utero

    ( a ) FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or 3 as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ (mean ± s.e.m.). ***p<0.001; *p<0.05, P values: Rap1GAP (RG): 9.8E-8, RG+FGFR1: 7.0E-4, RG+FGFR2: 3.0E-4, RG+FGFR3: 0.020 ( n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3). ( b ) Protein abundance of FGFR1-GFP is regulated by Rap1 in vivo. E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP and either vector or pNeuroD-Rap1GAP. Two days later, mCherry and FGFR1-GFP were detected by epifluorescence. The graphs show mean and standard deviation of image intensity measured across lines drawn through the center of the cell body for eight neurons in each case. ( c ) Embryonic cortical neurons were electroporated to overexpress pCAG-NCad-HA or with a control plasmid, cultured for 2 days then analyzed for the protein level of NCad-HA and endogenous FGFR1 by Western blot. 10.7554/eLife.47673.009 Figure 3—source data 1. FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or three as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Table shows the percentage of cells in the RMZ. (n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3).
    Figure Legend Snippet: ( a ) FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or 3 as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ (mean ± s.e.m.). ***p<0.001; *p<0.05, P values: Rap1GAP (RG): 9.8E-8, RG+FGFR1: 7.0E-4, RG+FGFR2: 3.0E-4, RG+FGFR3: 0.020 ( n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3). ( b ) Protein abundance of FGFR1-GFP is regulated by Rap1 in vivo. E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP and either vector or pNeuroD-Rap1GAP. Two days later, mCherry and FGFR1-GFP were detected by epifluorescence. The graphs show mean and standard deviation of image intensity measured across lines drawn through the center of the cell body for eight neurons in each case. ( c ) Embryonic cortical neurons were electroporated to overexpress pCAG-NCad-HA or with a control plasmid, cultured for 2 days then analyzed for the protein level of NCad-HA and endogenous FGFR1 by Western blot. 10.7554/eLife.47673.009 Figure 3—source data 1. FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or three as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Table shows the percentage of cells in the RMZ. (n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3).

    Techniques Used: Migration, Inhibition, In Utero, Plasmid Preparation, Labeling, In Vivo, Standard Deviation, Cell Culture, Western Blot

    ( a ) Cells were transfected with pCAG-NCad-HA, ECad-HA or vector together with pCAG-FGFR1, 2 or 3-Myc and protein abundance determined by Western blot 24 hr later. ( b ) FGFR1(DN) does not change NCad protein level and does not reduce NCad homophilic interaction. Cells were transfected with pCAG-NCad-Myc, pCAG-NCad-HA and pCAG-FGFR1(DN)-GFP or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad-HA accumulates at cell-cell junctions in the presence of FGFR(DN)-GFP. Immunostaining of cells expressing NCad-HA alone of with FGFR(DN)-GFP. Arrows show NCad-HA at cell-cell junctions.
    Figure Legend Snippet: ( a ) Cells were transfected with pCAG-NCad-HA, ECad-HA or vector together with pCAG-FGFR1, 2 or 3-Myc and protein abundance determined by Western blot 24 hr later. ( b ) FGFR1(DN) does not change NCad protein level and does not reduce NCad homophilic interaction. Cells were transfected with pCAG-NCad-Myc, pCAG-NCad-HA and pCAG-FGFR1(DN)-GFP or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad-HA accumulates at cell-cell junctions in the presence of FGFR(DN)-GFP. Immunostaining of cells expressing NCad-HA alone of with FGFR(DN)-GFP. Arrows show NCad-HA at cell-cell junctions.

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

    ( a ) Cells were transfected separately to express NCad-GFP ( B, D and F ), NCad-HA ( A ), NCad W161A -HA ( C ), or vector ( E ). Twice the amount of DNA was used for NCad W161A -HA transfection to equalize the protein level with NCad-HA. After transfection, cells were mixed as described in the table and re-plated allowing only trans interactions to be detected. 24 hr later, cells were lysed, immunoprecipitated with anti-HA antibody, and proteins detected by Western blot. ( b ) Cells were co-transfected with NCad-GFP and either NCad-HA, NCad W161A -HA or vector. Cells were then left in contact or kept in suspension to prevent cell contact and enable cis interactions only. Receptor interactions were then tested by co-immunoprecipitation as above. ( c ) NCad W161A increased the protein abundance of FGFR1 as efficiently as wild-type NCad. 293T cells were transfected for the expression of the indicated proteins. Twice the amount of DNA was used for NCad W161A in order to equalize protein levels. Cell lysates were analyzed by Western blot. All experiments were repeated three times with similar results.
    Figure Legend Snippet: ( a ) Cells were transfected separately to express NCad-GFP ( B, D and F ), NCad-HA ( A ), NCad W161A -HA ( C ), or vector ( E ). Twice the amount of DNA was used for NCad W161A -HA transfection to equalize the protein level with NCad-HA. After transfection, cells were mixed as described in the table and re-plated allowing only trans interactions to be detected. 24 hr later, cells were lysed, immunoprecipitated with anti-HA antibody, and proteins detected by Western blot. ( b ) Cells were co-transfected with NCad-GFP and either NCad-HA, NCad W161A -HA or vector. Cells were then left in contact or kept in suspension to prevent cell contact and enable cis interactions only. Receptor interactions were then tested by co-immunoprecipitation as above. ( c ) NCad W161A increased the protein abundance of FGFR1 as efficiently as wild-type NCad. 293T cells were transfected for the expression of the indicated proteins. Twice the amount of DNA was used for NCad W161A in order to equalize protein levels. Cell lysates were analyzed by Western blot. All experiments were repeated three times with similar results.

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

    ( a ) NCad EC4 is required for FGFR1 binding in vitro. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. To equalize FGFR1-Myc expression, half the amount of FGFR1-Myc was transfected with wildtype NCad. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( b ) EC4 is dispensable for NCad homophilic binding. Cells were transfected with pCAG-NCad-FLAG and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad increases FGFR protein level dependent on EC4, and increases FGFR and Erk1/2 phosphorylation dependent on EC4 and FGFR kinase activity. HEK293T cells were transfected with equal amounts of pCAG-FGFR1-Myc DNA and either pCAG-NCad-HA, pCAG-NCad ΔEC4 -HA or vector. 24 hr after transfection, the specific FGFR inhibitor Debio1347 was used at 5 µM for 2 hr. Lysates were analyzed by Western blot using the indicated antibodies. Experiments a–c ) were repeated independently three times with similar results. ( d ) NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCad ΔEC4 -HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The graph shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCad ΔEC4 . P value: 0.116. Scale bar 100 µm. Error bars, s.e.m., NS, not significant. 10.7554/eLife.47673.016 Figure 5—source data 1. NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCadDEC4-HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The table shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCadDEC4.
    Figure Legend Snippet: ( a ) NCad EC4 is required for FGFR1 binding in vitro. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. To equalize FGFR1-Myc expression, half the amount of FGFR1-Myc was transfected with wildtype NCad. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( b ) EC4 is dispensable for NCad homophilic binding. Cells were transfected with pCAG-NCad-FLAG and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad increases FGFR protein level dependent on EC4, and increases FGFR and Erk1/2 phosphorylation dependent on EC4 and FGFR kinase activity. HEK293T cells were transfected with equal amounts of pCAG-FGFR1-Myc DNA and either pCAG-NCad-HA, pCAG-NCad ΔEC4 -HA or vector. 24 hr after transfection, the specific FGFR inhibitor Debio1347 was used at 5 µM for 2 hr. Lysates were analyzed by Western blot using the indicated antibodies. Experiments a–c ) were repeated independently three times with similar results. ( d ) NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCad ΔEC4 -HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The graph shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCad ΔEC4 . P value: 0.116. Scale bar 100 µm. Error bars, s.e.m., NS, not significant. 10.7554/eLife.47673.016 Figure 5—source data 1. NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCadDEC4-HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The table shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCadDEC4.

    Techniques Used: Binding Assay, In Vitro, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Activity Assay, Migration, In Utero, Labeling

    ( a ) FGFR1-NCad binding does not occur in trans, between cells. Cells were transfected with FGFR1-Myc and NCad-HA together ( A ), NCad-HA and vector ( C ), or FGFR1-Myc and vector ( D and F ). The transfected cells were removed from the dish, mixed as shown, and re-plated to allow cell-cell contact. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA when co-expressed ( A ) but not when binding could only occur between cells (C+D). ( b ) FGFR1-NCad binding occurs in cis, within cells. Cells were transfected with FGFR1-Myc and NCad-HA or vector, then left in contact or placed in suspension to prevent cell-cell interaction. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA whether or not cell-cell contact was permitted. All experiments were repeated three times with similar results.
    Figure Legend Snippet: ( a ) FGFR1-NCad binding does not occur in trans, between cells. Cells were transfected with FGFR1-Myc and NCad-HA together ( A ), NCad-HA and vector ( C ), or FGFR1-Myc and vector ( D and F ). The transfected cells were removed from the dish, mixed as shown, and re-plated to allow cell-cell contact. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA when co-expressed ( A ) but not when binding could only occur between cells (C+D). ( b ) FGFR1-NCad binding occurs in cis, within cells. Cells were transfected with FGFR1-Myc and NCad-HA or vector, then left in contact or placed in suspension to prevent cell-cell interaction. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA whether or not cell-cell contact was permitted. All experiments were repeated three times with similar results.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    24 hr later, cells were lysed and lysates and anti-HA immunoprecipitates analyzed by Western blot. To equalize FGFR1-Myc expression level, half the amount of FGFR1-Myc DNA was transfected with NCad-HA or NCad W161A -HA.
    Figure Legend Snippet: 24 hr later, cells were lysed and lysates and anti-HA immunoprecipitates analyzed by Western blot. To equalize FGFR1-Myc expression level, half the amount of FGFR1-Myc DNA was transfected with NCad-HA or NCad W161A -HA.

    Techniques Used: Western Blot, Expressing, Transfection

    ( a ) NCad but not NCad ΔEC4 inhibits FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad, NCad ΔEC4 -HA or vector. One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and ubiquitin. To equalize FGFR1-Myc levels, half the amount of DNA was used for FGFR1-Myc when expressed with NCad-HA. ( b ) HA-Ubi KO but not HA-Ubi WT increased FGFR1 protein level and decreased FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and HA-Ubi KO or HA-Ubi WT . One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and HA. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The graph shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). P values: Ubi K6R : 0.026, Ubi K11R : 0.034, Ubi K27R : 0.490, Ubi K29R : 0.466, Ubi K33R : 0.036, Ubi K48R : 0.032, Ubi K63R : 0.024, Ubi WT : 4.8E-4. n = 4 Ubi K6R , 3 Ubi K11R , 3 Ubi K27R , 3 Ubi K29R , 4 Ubi K33R , 4 Ubi K48R , 3 Ubi K63R , 8 Ubi WT . ( d ) FGFR1 levels remain normal only when both K27- and K29-linked polyubiquitination are permitted. HA-Ubiquitin mutants in which all but one lysine is mutated into arginine were used to allow only one type of polyubiquitin chain formation (Ubi K27 and Ubi K29 ). ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The graph shows the percentage of cells in the RMZ (mean ± s.e.m.). P values: RG+Ubi K27R : 1.7E-3, RG+Ubi WT : 0.471. n = 4 Rap1GAP (RG), 8 RG+Ubi K27R , 6 RG+Ubi WT . ( f ) Endogenous FGFR1 is degraded by the lysosome in vivo. Primary embryonic cortical neurons were cultured in the presence of 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Similar results were obtained in three independent experiments. Scale bar 100 µm. *p<0.05, **p<0.01 ***p<0.001, NS not significant. 10.7554/eLife.47673.022 Figure 7—source data 1. Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level and rescues the migration defect of Rap1GAP-expressing cells. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The table shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). n = 4 UbiK6R, 3 UbiK11R, 3 UbiK27R, 3 UbiK29R, 4 UbiK33R, 4 UbiK48R, 3 UbiK63R, 8 UbiWT. ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The table shows the percentage of cells in the RMZ (mean ± s.e.m.). n = 4 Rap1GAP (RG), 8 RG+UbiK27R, 6 RG+UbiWT.
    Figure Legend Snippet: ( a ) NCad but not NCad ΔEC4 inhibits FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad, NCad ΔEC4 -HA or vector. One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and ubiquitin. To equalize FGFR1-Myc levels, half the amount of DNA was used for FGFR1-Myc when expressed with NCad-HA. ( b ) HA-Ubi KO but not HA-Ubi WT increased FGFR1 protein level and decreased FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and HA-Ubi KO or HA-Ubi WT . One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and HA. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The graph shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). P values: Ubi K6R : 0.026, Ubi K11R : 0.034, Ubi K27R : 0.490, Ubi K29R : 0.466, Ubi K33R : 0.036, Ubi K48R : 0.032, Ubi K63R : 0.024, Ubi WT : 4.8E-4. n = 4 Ubi K6R , 3 Ubi K11R , 3 Ubi K27R , 3 Ubi K29R , 4 Ubi K33R , 4 Ubi K48R , 3 Ubi K63R , 8 Ubi WT . ( d ) FGFR1 levels remain normal only when both K27- and K29-linked polyubiquitination are permitted. HA-Ubiquitin mutants in which all but one lysine is mutated into arginine were used to allow only one type of polyubiquitin chain formation (Ubi K27 and Ubi K29 ). ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The graph shows the percentage of cells in the RMZ (mean ± s.e.m.). P values: RG+Ubi K27R : 1.7E-3, RG+Ubi WT : 0.471. n = 4 Rap1GAP (RG), 8 RG+Ubi K27R , 6 RG+Ubi WT . ( f ) Endogenous FGFR1 is degraded by the lysosome in vivo. Primary embryonic cortical neurons were cultured in the presence of 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Similar results were obtained in three independent experiments. Scale bar 100 µm. *p<0.05, **p<0.01 ***p<0.001, NS not significant. 10.7554/eLife.47673.022 Figure 7—source data 1. Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level and rescues the migration defect of Rap1GAP-expressing cells. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The table shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). n = 4 UbiK6R, 3 UbiK11R, 3 UbiK27R, 3 UbiK29R, 4 UbiK33R, 4 UbiK48R, 3 UbiK63R, 8 UbiWT. ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The table shows the percentage of cells in the RMZ (mean ± s.e.m.). n = 4 Rap1GAP (RG), 8 RG+UbiK27R, 6 RG+UbiWT.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Inhibition, Conjugation Assay, Mutagenesis, Expressing, In Vivo, Migration, In Utero, Electroporation, Cell Culture

    Cells co-transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA or vector were incubated with 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Epoxomycin inhibited proteasomal degradation of many ubiquitinated cell proteins, as indicated by an increase in total protein ubiquitination.
    Figure Legend Snippet: Cells co-transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA or vector were incubated with 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Epoxomycin inhibited proteasomal degradation of many ubiquitinated cell proteins, as indicated by an increase in total protein ubiquitination.

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

    E16.5 mouse cortical neurons were cultured for 3 days then stimulated with FGF2 or Reelin for different times. All experiments were repeated three times with similar results. ( a ) Short-term Reelin stimulation does not increase FGFR1 protein level or Erk1/2 phosphorylation. Neurons were stimulated for 20 min with 75 ng/ml FGF2, Mock- or Reelin-conditioned media. ( b ) Long-term Reelin stimulation increases FGFR1 protein level and FGFR1 and Erk1/2 phosphorylation dependent on FGFR1 kinase activity. Neurons were stimulated for 15 hr with Mock- or Reelin-conditioned media or for 20 min with FGF2 or EGF. FGFR inhibitor Debio1347 was used at a concentration of 5 µM for a total of 17 hr before cell lysis. ( c ) Reelin fragment R3-6 induces FGFR1 accumulation and Erk1/2 activation. Neurons were stimulated for 15 hr with Mock, R3-6 or Reelin-conditioned medium. ( d ) Long-term Reelin stimulation of FGFR1 protein l evel and Erk phosphorylation requires Rap1 and NCad. Neurons were electroporated with pCAG-Rap1GAP, pCAG-NCad DN , or vector, incubated for 2 days, then stimulated with Mock- or Reelin-conditioned media for 15 hr and analyzed by Western blotting.
    Figure Legend Snippet: E16.5 mouse cortical neurons were cultured for 3 days then stimulated with FGF2 or Reelin for different times. All experiments were repeated three times with similar results. ( a ) Short-term Reelin stimulation does not increase FGFR1 protein level or Erk1/2 phosphorylation. Neurons were stimulated for 20 min with 75 ng/ml FGF2, Mock- or Reelin-conditioned media. ( b ) Long-term Reelin stimulation increases FGFR1 protein level and FGFR1 and Erk1/2 phosphorylation dependent on FGFR1 kinase activity. Neurons were stimulated for 15 hr with Mock- or Reelin-conditioned media or for 20 min with FGF2 or EGF. FGFR inhibitor Debio1347 was used at a concentration of 5 µM for a total of 17 hr before cell lysis. ( c ) Reelin fragment R3-6 induces FGFR1 accumulation and Erk1/2 activation. Neurons were stimulated for 15 hr with Mock, R3-6 or Reelin-conditioned medium. ( d ) Long-term Reelin stimulation of FGFR1 protein l evel and Erk phosphorylation requires Rap1 and NCad. Neurons were electroporated with pCAG-Rap1GAP, pCAG-NCad DN , or vector, incubated for 2 days, then stimulated with Mock- or Reelin-conditioned media for 15 hr and analyzed by Western blotting.

    Techniques Used: Cell Culture, Activity Assay, Concentration Assay, Lysis, Activation Assay, Plasmid Preparation, Incubation, Western Blot


    Figure Legend Snippet:

    Techniques Used: FLAG-tag, Recombinant, Sequencing, Protease Inhibitor, Plasmid Preparation, DNA Purification, Gel Extraction, Software, Staining, In Vitro, Transfection

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    ( a,b ) shRNA against <t>FGFR1,</t> 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.
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    Article Snippet: Rabbit monoclonal FGFR1 antibody D8E4 , Cell Signaling Technology , 9740.

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    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Small-molecule and CRISPR screening converge to reveal RTK dependencies in pediatric rhabdoid tumors

    doi: 10.1016/j.celrep.2019.07.021

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: FGFR1 (D8E4) , Cell Signaling , Cat#9740; RRID: AB_11178519.

    Techniques: Recombinant, Cell Viability Assay, Protease Inhibitor, Injection, Sample Prep, Expressing, Activity Assay, Plasmid Preparation, Software, Methylation

    Journal: eLife

    Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

    doi: 10.7554/eLife.52027

    Figure Lengend Snippet:

    Article Snippet: Antibody , anti-FGFR1 (monoclonal polyclonal) , Cell Signaling Technology D8E4 9740 , RRID: AB_11178519 , WB (1:1000).

    Techniques: Transfection, Construct, Clone Assay, Plasmid Preparation, Electroporation, shRNA, Recombinant, Sequencing, Immunoprecipitation, Software

    ( a,b ) shRNA against FGFR1, 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a,b ) shRNA against FGFR1, 2 or 3 and dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. ( a ) pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and ( b ) shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day ( E ) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3. P values: FGFR1(DN): 9.6E-6, FGFR2(DN): 4.2E-6, FGFR3(DN): 4.0E-5, FGFR1: 0.245, FGFR2: 0.170, FGFR3: 0.353, shFGFR1: 3.0E-4, shFGFR2: 2.9E-3, shFGFR3: 0.169, shFGFR1+2: 6.4E-5, shFGFR1+3: 1.0E-4, shFGFR2+3: 3.2E-3. Error bars, s.e.m. ***p<0.001, **p<0.01, *p<0.05, NS, not significant Scale bar 100 µm. 10.7554/eLife.47673.004 Figure 1—source data 1. FGFRs regulate projection neuron migration in vivo. ( a,b ) shRNA against FGFR1, 2 or three and Dominant-negative (DN) but not wildtype FGFR1, 2 or 3 induce an accumulation of neurons at the MMZ. a pNeuroD-FGFR(DN) and pNeuroD-FGFR plasmids, expressed in neurons, and b shRNA against FGFR1, 2 or 3 or negative control shRNA (shCtrl) were co-electroporated in utero with pCAG-GFP, expressed in progenitors and neurons, at embryonic day (E) 14.5. ( a,b ) Three days later, cryosections were prepared and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Tables show the percentage of cells in the RMZ. n = 5 Control, 7 FGFR1(DN), 4 FGFR2(DN), 4 FGFR3(DN), 3 FGFR1, 3 FGFR2, 4 FGFR3, 6 shCtrl, 4 shFGFR1, 4 shFGFR2, 4 shFGFR3, 5 shFGFR1+2, 3 shFGFR1+3, 3 shFGFR2+3.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: shRNA, Dominant Negative Mutation, Negative Control, In Utero, Labeling, Migration, In Vivo

    ( a ) HEK293T cells were transfected with pCAG-FGFR1-GFP, FGFR2-GFP, FGFR3-GFP together with expression vectors for shRNAs against the corresponding receptor or a negative control shRNA (shCtrl). Protein levels were determined by Western blot two days later. ( b ) E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP, FGFR2-GFP or FGFR3-GFP and an expression vectors for shRNA against the corresponding receptor or negative control shRNA. Two days later, CherryFP and FGFRs-GFP were detected by epifluorescence.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) HEK293T cells were transfected with pCAG-FGFR1-GFP, FGFR2-GFP, FGFR3-GFP together with expression vectors for shRNAs against the corresponding receptor or a negative control shRNA (shCtrl). Protein levels were determined by Western blot two days later. ( b ) E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP, FGFR2-GFP or FGFR3-GFP and an expression vectors for shRNA against the corresponding receptor or negative control shRNA. Two days later, CherryFP and FGFRs-GFP were detected by epifluorescence.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Transfection, Expressing, Negative Control, shRNA, Western Blot, In Utero

    ( a ) FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or 3 as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ (mean ± s.e.m.). ***p<0.001; *p<0.05, P values: Rap1GAP (RG): 9.8E-8, RG+FGFR1: 7.0E-4, RG+FGFR2: 3.0E-4, RG+FGFR3: 0.020 ( n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3). ( b ) Protein abundance of FGFR1-GFP is regulated by Rap1 in vivo. E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP and either vector or pNeuroD-Rap1GAP. Two days later, mCherry and FGFR1-GFP were detected by epifluorescence. The graphs show mean and standard deviation of image intensity measured across lines drawn through the center of the cell body for eight neurons in each case. ( c ) Embryonic cortical neurons were electroporated to overexpress pCAG-NCad-HA or with a control plasmid, cultured for 2 days then analyzed for the protein level of NCad-HA and endogenous FGFR1 by Western blot. 10.7554/eLife.47673.009 Figure 3—source data 1. FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or three as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Table shows the percentage of cells in the RMZ. (n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3).

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or 3 as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Graphs show the percentage of cells in the RMZ (mean ± s.e.m.). ***p<0.001; *p<0.05, P values: Rap1GAP (RG): 9.8E-8, RG+FGFR1: 7.0E-4, RG+FGFR2: 3.0E-4, RG+FGFR3: 0.020 ( n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3). ( b ) Protein abundance of FGFR1-GFP is regulated by Rap1 in vivo. E14.5 embryos were electroporated in utero with a mixture of pCAG-CherryFP, pNeuroD-FGFR1-GFP and either vector or pNeuroD-Rap1GAP. Two days later, mCherry and FGFR1-GFP were detected by epifluorescence. The graphs show mean and standard deviation of image intensity measured across lines drawn through the center of the cell body for eight neurons in each case. ( c ) Embryonic cortical neurons were electroporated to overexpress pCAG-NCad-HA or with a control plasmid, cultured for 2 days then analyzed for the protein level of NCad-HA and endogenous FGFR1 by Western blot. 10.7554/eLife.47673.009 Figure 3—source data 1. FGFR1, 2 and 3 partially rescue the neuronal migration phenotype induced by Rap1 inhibition. E14.5 embryos were electroporated in utero with pCAG-GFP, pNeuroD vector or pNeuroD-Rap1GAP (RG), and pNeuroD-FGFR1, 2 or three as shown. Cryosections were prepared 3 days later and labeled for DAPI (blue) and GFP (green). The cerebral wall was subdivided into radial morphology zone (RMZ), multipolar morphology zone (MMZ) and VZ. Table shows the percentage of cells in the RMZ. (n = 4 Control, 4 Rap1GAP (RG), 7 RG+FGFR1, 7 RG+FGFR2, 4 RG+FGFR3).

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Migration, Inhibition, In Utero, Plasmid Preparation, Labeling, In Vivo, Standard Deviation, Cell Culture, Western Blot

    ( a ) Cells were transfected with pCAG-NCad-HA, ECad-HA or vector together with pCAG-FGFR1, 2 or 3-Myc and protein abundance determined by Western blot 24 hr later. ( b ) FGFR1(DN) does not change NCad protein level and does not reduce NCad homophilic interaction. Cells were transfected with pCAG-NCad-Myc, pCAG-NCad-HA and pCAG-FGFR1(DN)-GFP or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad-HA accumulates at cell-cell junctions in the presence of FGFR(DN)-GFP. Immunostaining of cells expressing NCad-HA alone of with FGFR(DN)-GFP. Arrows show NCad-HA at cell-cell junctions.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) Cells were transfected with pCAG-NCad-HA, ECad-HA or vector together with pCAG-FGFR1, 2 or 3-Myc and protein abundance determined by Western blot 24 hr later. ( b ) FGFR1(DN) does not change NCad protein level and does not reduce NCad homophilic interaction. Cells were transfected with pCAG-NCad-Myc, pCAG-NCad-HA and pCAG-FGFR1(DN)-GFP or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad-HA accumulates at cell-cell junctions in the presence of FGFR(DN)-GFP. Immunostaining of cells expressing NCad-HA alone of with FGFR(DN)-GFP. Arrows show NCad-HA at cell-cell junctions.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Immunostaining, Expressing

    ( a ) Cells were transfected separately to express NCad-GFP ( B, D and F ), NCad-HA ( A ), NCad W161A -HA ( C ), or vector ( E ). Twice the amount of DNA was used for NCad W161A -HA transfection to equalize the protein level with NCad-HA. After transfection, cells were mixed as described in the table and re-plated allowing only trans interactions to be detected. 24 hr later, cells were lysed, immunoprecipitated with anti-HA antibody, and proteins detected by Western blot. ( b ) Cells were co-transfected with NCad-GFP and either NCad-HA, NCad W161A -HA or vector. Cells were then left in contact or kept in suspension to prevent cell contact and enable cis interactions only. Receptor interactions were then tested by co-immunoprecipitation as above. ( c ) NCad W161A increased the protein abundance of FGFR1 as efficiently as wild-type NCad. 293T cells were transfected for the expression of the indicated proteins. Twice the amount of DNA was used for NCad W161A in order to equalize protein levels. Cell lysates were analyzed by Western blot. All experiments were repeated three times with similar results.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) Cells were transfected separately to express NCad-GFP ( B, D and F ), NCad-HA ( A ), NCad W161A -HA ( C ), or vector ( E ). Twice the amount of DNA was used for NCad W161A -HA transfection to equalize the protein level with NCad-HA. After transfection, cells were mixed as described in the table and re-plated allowing only trans interactions to be detected. 24 hr later, cells were lysed, immunoprecipitated with anti-HA antibody, and proteins detected by Western blot. ( b ) Cells were co-transfected with NCad-GFP and either NCad-HA, NCad W161A -HA or vector. Cells were then left in contact or kept in suspension to prevent cell contact and enable cis interactions only. Receptor interactions were then tested by co-immunoprecipitation as above. ( c ) NCad W161A increased the protein abundance of FGFR1 as efficiently as wild-type NCad. 293T cells were transfected for the expression of the indicated proteins. Twice the amount of DNA was used for NCad W161A in order to equalize protein levels. Cell lysates were analyzed by Western blot. All experiments were repeated three times with similar results.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

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

    ( a ) NCad EC4 is required for FGFR1 binding in vitro. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. To equalize FGFR1-Myc expression, half the amount of FGFR1-Myc was transfected with wildtype NCad. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( b ) EC4 is dispensable for NCad homophilic binding. Cells were transfected with pCAG-NCad-FLAG and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad increases FGFR protein level dependent on EC4, and increases FGFR and Erk1/2 phosphorylation dependent on EC4 and FGFR kinase activity. HEK293T cells were transfected with equal amounts of pCAG-FGFR1-Myc DNA and either pCAG-NCad-HA, pCAG-NCad ΔEC4 -HA or vector. 24 hr after transfection, the specific FGFR inhibitor Debio1347 was used at 5 µM for 2 hr. Lysates were analyzed by Western blot using the indicated antibodies. Experiments a–c ) were repeated independently three times with similar results. ( d ) NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCad ΔEC4 -HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The graph shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCad ΔEC4 . P value: 0.116. Scale bar 100 µm. Error bars, s.e.m., NS, not significant. 10.7554/eLife.47673.016 Figure 5—source data 1. NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCadDEC4-HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The table shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCadDEC4.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) NCad EC4 is required for FGFR1 binding in vitro. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. To equalize FGFR1-Myc expression, half the amount of FGFR1-Myc was transfected with wildtype NCad. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( b ) EC4 is dispensable for NCad homophilic binding. Cells were transfected with pCAG-NCad-FLAG and pCAG-NCad-HA, NCad ΔEC4 -HA or vector. One day later, cells were lysed and immunoprecipitated with anti-HA. Lysates and co-immunoprecipitated proteins were analyzed by Western blot. ( c ) NCad increases FGFR protein level dependent on EC4, and increases FGFR and Erk1/2 phosphorylation dependent on EC4 and FGFR kinase activity. HEK293T cells were transfected with equal amounts of pCAG-FGFR1-Myc DNA and either pCAG-NCad-HA, pCAG-NCad ΔEC4 -HA or vector. 24 hr after transfection, the specific FGFR inhibitor Debio1347 was used at 5 µM for 2 hr. Lysates were analyzed by Western blot using the indicated antibodies. Experiments a–c ) were repeated independently three times with similar results. ( d ) NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCad ΔEC4 -HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The graph shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCad ΔEC4 . P value: 0.116. Scale bar 100 µm. Error bars, s.e.m., NS, not significant. 10.7554/eLife.47673.016 Figure 5—source data 1. NCad EC4 is required for the multipolar migration. E14.5 embryos were electroporated in utero with pCAG-GFP and pNeuroD-Rap1GAP (RG), pNeuroD-NCadDEC4-HA or vector. Cryosections were prepared three days later and labeled for DAPI (blue) and GFP (green). The table shows the percentage of cells in the RMZ. n = 4 control, 4 Rap1GAP (RG), 6 RG+ NCadDEC4.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Binding Assay, In Vitro, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Activity Assay, Migration, In Utero, Labeling

    ( a ) FGFR1-NCad binding does not occur in trans, between cells. Cells were transfected with FGFR1-Myc and NCad-HA together ( A ), NCad-HA and vector ( C ), or FGFR1-Myc and vector ( D and F ). The transfected cells were removed from the dish, mixed as shown, and re-plated to allow cell-cell contact. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA when co-expressed ( A ) but not when binding could only occur between cells (C+D). ( b ) FGFR1-NCad binding occurs in cis, within cells. Cells were transfected with FGFR1-Myc and NCad-HA or vector, then left in contact or placed in suspension to prevent cell-cell interaction. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA whether or not cell-cell contact was permitted. All experiments were repeated three times with similar results.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) FGFR1-NCad binding does not occur in trans, between cells. Cells were transfected with FGFR1-Myc and NCad-HA together ( A ), NCad-HA and vector ( C ), or FGFR1-Myc and vector ( D and F ). The transfected cells were removed from the dish, mixed as shown, and re-plated to allow cell-cell contact. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA when co-expressed ( A ) but not when binding could only occur between cells (C+D). ( b ) FGFR1-NCad binding occurs in cis, within cells. Cells were transfected with FGFR1-Myc and NCad-HA or vector, then left in contact or placed in suspension to prevent cell-cell interaction. One day later the cells were lysed and immunoprecipitated with anti-HA. Lysates and immunoprecipitates were analyzed by Western blotting. FGFR1-Myc co-precipitated with NCad-HA whether or not cell-cell contact was permitted. All experiments were repeated three times with similar results.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    24 hr later, cells were lysed and lysates and anti-HA immunoprecipitates analyzed by Western blot. To equalize FGFR1-Myc expression level, half the amount of FGFR1-Myc DNA was transfected with NCad-HA or NCad W161A -HA.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: 24 hr later, cells were lysed and lysates and anti-HA immunoprecipitates analyzed by Western blot. To equalize FGFR1-Myc expression level, half the amount of FGFR1-Myc DNA was transfected with NCad-HA or NCad W161A -HA.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Western Blot, Expressing, Transfection

    ( a ) NCad but not NCad ΔEC4 inhibits FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad, NCad ΔEC4 -HA or vector. One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and ubiquitin. To equalize FGFR1-Myc levels, half the amount of DNA was used for FGFR1-Myc when expressed with NCad-HA. ( b ) HA-Ubi KO but not HA-Ubi WT increased FGFR1 protein level and decreased FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and HA-Ubi KO or HA-Ubi WT . One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and HA. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The graph shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). P values: Ubi K6R : 0.026, Ubi K11R : 0.034, Ubi K27R : 0.490, Ubi K29R : 0.466, Ubi K33R : 0.036, Ubi K48R : 0.032, Ubi K63R : 0.024, Ubi WT : 4.8E-4. n = 4 Ubi K6R , 3 Ubi K11R , 3 Ubi K27R , 3 Ubi K29R , 4 Ubi K33R , 4 Ubi K48R , 3 Ubi K63R , 8 Ubi WT . ( d ) FGFR1 levels remain normal only when both K27- and K29-linked polyubiquitination are permitted. HA-Ubiquitin mutants in which all but one lysine is mutated into arginine were used to allow only one type of polyubiquitin chain formation (Ubi K27 and Ubi K29 ). ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The graph shows the percentage of cells in the RMZ (mean ± s.e.m.). P values: RG+Ubi K27R : 1.7E-3, RG+Ubi WT : 0.471. n = 4 Rap1GAP (RG), 8 RG+Ubi K27R , 6 RG+Ubi WT . ( f ) Endogenous FGFR1 is degraded by the lysosome in vivo. Primary embryonic cortical neurons were cultured in the presence of 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Similar results were obtained in three independent experiments. Scale bar 100 µm. *p<0.05, **p<0.01 ***p<0.001, NS not significant. 10.7554/eLife.47673.022 Figure 7—source data 1. Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level and rescues the migration defect of Rap1GAP-expressing cells. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The table shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). n = 4 UbiK6R, 3 UbiK11R, 3 UbiK27R, 3 UbiK29R, 4 UbiK33R, 4 UbiK48R, 3 UbiK63R, 8 UbiWT. ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The table shows the percentage of cells in the RMZ (mean ± s.e.m.). n = 4 Rap1GAP (RG), 8 RG+UbiK27R, 6 RG+UbiWT.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: ( a ) NCad but not NCad ΔEC4 inhibits FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and pCAG-NCad, NCad ΔEC4 -HA or vector. One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and ubiquitin. To equalize FGFR1-Myc levels, half the amount of DNA was used for FGFR1-Myc when expressed with NCad-HA. ( b ) HA-Ubi KO but not HA-Ubi WT increased FGFR1 protein level and decreased FGFR1 ubiquitination. Cells were transfected with pCAG-FGFR1-Myc and HA-Ubi KO or HA-Ubi WT . One day later, cells were lysed and proteins immunoprecipitated with anti-Myc. Lysates and immunoprecipitates were analyzed with Western blotting using antibodies to Myc and HA. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The graph shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). P values: Ubi K6R : 0.026, Ubi K11R : 0.034, Ubi K27R : 0.490, Ubi K29R : 0.466, Ubi K33R : 0.036, Ubi K48R : 0.032, Ubi K63R : 0.024, Ubi WT : 4.8E-4. n = 4 Ubi K6R , 3 Ubi K11R , 3 Ubi K27R , 3 Ubi K29R , 4 Ubi K33R , 4 Ubi K48R , 3 Ubi K63R , 8 Ubi WT . ( d ) FGFR1 levels remain normal only when both K27- and K29-linked polyubiquitination are permitted. HA-Ubiquitin mutants in which all but one lysine is mutated into arginine were used to allow only one type of polyubiquitin chain formation (Ubi K27 and Ubi K29 ). ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The graph shows the percentage of cells in the RMZ (mean ± s.e.m.). P values: RG+Ubi K27R : 1.7E-3, RG+Ubi WT : 0.471. n = 4 Rap1GAP (RG), 8 RG+Ubi K27R , 6 RG+Ubi WT . ( f ) Endogenous FGFR1 is degraded by the lysosome in vivo. Primary embryonic cortical neurons were cultured in the presence of 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Similar results were obtained in three independent experiments. Scale bar 100 µm. *p<0.05, **p<0.01 ***p<0.001, NS not significant. 10.7554/eLife.47673.022 Figure 7—source data 1. Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level and rescues the migration defect of Rap1GAP-expressing cells. ( c ) Inhibition of K27- and K29-linked polyubiquitination increases FGFR1 protein level. Ubiquitin-GFP mutants in which one lysine is mutated into arginine were used to identify lysine residues required for polyubiquitin chain formation. Co-translational cleavage detaches the GFP and frees the terminal glycine of ubiquitin for subsequent conjugation . The cleaved GFP was used to quantify ubiquitin mutant expression. The table shows the relative FGFR1-Myc band intensity when expressed in the presence or absence of NCad-HA and in the presence of an ubiquitin mutant as indicated (mean ± s.e.m.). n = 4 UbiK6R, 3 UbiK11R, 3 UbiK27R, 3 UbiK29R, 4 UbiK33R, 4 UbiK48R, 3 UbiK63R, 8 UbiWT. ( e ) Inhibition of K27-linked polyubiquitin chain formation in vivo rescues the migration defect of Rap1GAP-expressing cells. In utero electroporation at embryonic day E14.5 and analysis 3 days later. Plasmids coding for the indicated proteins and GFP were co-electroporated. The table shows the percentage of cells in the RMZ (mean ± s.e.m.). n = 4 Rap1GAP (RG), 8 RG+UbiK27R, 6 RG+UbiWT.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Inhibition, Conjugation Assay, Mutagenesis, Expressing, In Vivo, Migration, In Utero, Electroporation, Cell Culture

    Cells co-transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA or vector were incubated with 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Epoxomycin inhibited proteasomal degradation of many ubiquitinated cell proteins, as indicated by an increase in total protein ubiquitination.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: Cells co-transfected with pCAG-FGFR1-Myc and pCAG-NCad-HA or vector were incubated with 250 nM proteasome inhibitor epoxomycin or 300 µM lysosome inhibitor leupeptin for 4 hr and analyzed by Western blot. Epoxomycin inhibited proteasomal degradation of many ubiquitinated cell proteins, as indicated by an increase in total protein ubiquitination.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot

    E16.5 mouse cortical neurons were cultured for 3 days then stimulated with FGF2 or Reelin for different times. All experiments were repeated three times with similar results. ( a ) Short-term Reelin stimulation does not increase FGFR1 protein level or Erk1/2 phosphorylation. Neurons were stimulated for 20 min with 75 ng/ml FGF2, Mock- or Reelin-conditioned media. ( b ) Long-term Reelin stimulation increases FGFR1 protein level and FGFR1 and Erk1/2 phosphorylation dependent on FGFR1 kinase activity. Neurons were stimulated for 15 hr with Mock- or Reelin-conditioned media or for 20 min with FGF2 or EGF. FGFR inhibitor Debio1347 was used at a concentration of 5 µM for a total of 17 hr before cell lysis. ( c ) Reelin fragment R3-6 induces FGFR1 accumulation and Erk1/2 activation. Neurons were stimulated for 15 hr with Mock, R3-6 or Reelin-conditioned medium. ( d ) Long-term Reelin stimulation of FGFR1 protein l evel and Erk phosphorylation requires Rap1 and NCad. Neurons were electroporated with pCAG-Rap1GAP, pCAG-NCad DN , or vector, incubated for 2 days, then stimulated with Mock- or Reelin-conditioned media for 15 hr and analyzed by Western blotting.

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet: E16.5 mouse cortical neurons were cultured for 3 days then stimulated with FGF2 or Reelin for different times. All experiments were repeated three times with similar results. ( a ) Short-term Reelin stimulation does not increase FGFR1 protein level or Erk1/2 phosphorylation. Neurons were stimulated for 20 min with 75 ng/ml FGF2, Mock- or Reelin-conditioned media. ( b ) Long-term Reelin stimulation increases FGFR1 protein level and FGFR1 and Erk1/2 phosphorylation dependent on FGFR1 kinase activity. Neurons were stimulated for 15 hr with Mock- or Reelin-conditioned media or for 20 min with FGF2 or EGF. FGFR inhibitor Debio1347 was used at a concentration of 5 µM for a total of 17 hr before cell lysis. ( c ) Reelin fragment R3-6 induces FGFR1 accumulation and Erk1/2 activation. Neurons were stimulated for 15 hr with Mock, R3-6 or Reelin-conditioned medium. ( d ) Long-term Reelin stimulation of FGFR1 protein l evel and Erk phosphorylation requires Rap1 and NCad. Neurons were electroporated with pCAG-Rap1GAP, pCAG-NCad DN , or vector, incubated for 2 days, then stimulated with Mock- or Reelin-conditioned media for 15 hr and analyzed by Western blotting.

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: Cell Culture, Activity Assay, Concentration Assay, Lysis, Activation Assay, Plasmid Preparation, Incubation, Western Blot

    Journal: eLife

    Article Title: N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration

    doi: 10.7554/eLife.47673

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-FGFR1(D8E4) XP (Rabbit monoclonal) , Cell Signaling Technology , Cat# 9740, RRID: AB_11178519 , WB (1:500).

    Techniques: FLAG-tag, Recombinant, Sequencing, Protease Inhibitor, Plasmid Preparation, DNA Purification, Gel Extraction, Software, Staining, In Vitro, Transfection