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    Addgene inc paper addgene 174407 vamp4 kd2 mscarlet
    Figure 1. VAMP2 and <t>VAMP4</t> are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)
    Paper Addgene 174407 Vamp4 Kd2 Mscarlet, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/paper addgene 174407 vamp4 kd2 mscarlet/product/Addgene inc
    Average 93 stars, based on 15 article reviews
    paper addgene 174407 vamp4 kd2 mscarlet - by Bioz Stars, 2026-03
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    1) Product Images from "The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites."

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.109678

    Figure 1. VAMP2 and VAMP4 are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)
    Figure Legend Snippet: Figure 1. VAMP2 and VAMP4 are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)

    Techniques Used: Transfection

    Figure 2. Downregulation of VAMP4, but not VAMP2, impairs RE exocytosis and recycling to the plasma membrane (A) Frequency of exocytosis events in neurons transfected with TfR-SEP and TeNT-LC E234Q (n = 6) or TeNT-LC (n = 10). (B) Images of neurons co-transfected with VAMP2-SEP and TeNT-LC E234Q or TeNT-LC. VAMP2-SEP is enriched in the axon (cyan arrows) in the first case but not the second case. Scale bar, 10 mm. (C) Immunofluorescence images of endogenous VAMP4 in cells expressing GFP and a combination of shRNA targeted against VAMP4 for four days. In cells expressing GFP and the shRNA (cyan arrows), the labeling is strongly decreased compared to untransfected cells or cells expressing scramble (scr) shRNA. In cells co-expressing TfR, VAMP4-HA, and KD1, the VAMP4 staining is strong. Scale bar, 10 mm. Bottom, quantification of VAMP4 staining in the area delimited by the GFP mask (soma and dendrites). The staining is decreased by 50% in all KD conditions. The number of cells is indicated above the bars for all conditions. Comparison with scr with one-way ANOVA; *p < 0.05 and ***p < 0.001. (D) Frequency of exocytosis events recorded in cells expressing TfR-SEP and shRNAs targeted to VAMP4: scr (33 cells; 3 cells have frequencies of 0.132, 0.157, and 0.119 events.mm2.min1 and are represented above the axis limit), KD1 (23 cells), KD2 (10 cells), KD1+2 (18 cells), cells expressing VAMP4-HA (8 cells), and KD1+VAMP4-HA (12 cells). *p < 0.05 one-way ANOVA. (E) Images of neurons expressing scr or KD1 shRNAs in GFP vectors, labeled with A568-Tf (50 mg/ml) for 5 min and chased with unlabeled transferrin (2 mg/ml) at 37C for the indicated times. Scale bar, 10 mm. (F) Quantification of the Alexa568 fluorescence in the GFP mask from the pulse-chase experiments described in (E). 70 to 88 cells per condition from 4 inde- pendent experiments. Error bars represent SEM; **p < 0.01. (G) Estimation of TfR-SEP surface fraction. Top, cartoons showing the fraction of fluorescent TfR-SEP. At pH 7.4, surface receptors are fluorescent, but not at pH 5.5. Receptors in acidic intracellular organelles are not fluorescent, but become fluorescent with NH4Cl. Bottom left, images of a dendrite bathed successively in solutions at pH 7.4 (images 1, 3, and 5), pH 5.5 (image 2), and pH 7.4 containing NH4Cl (image 4). For image 4, the contrast is 23 lower than in the other images. Bottom right, quantification of the TfR-SEP surface fraction for neurons transfected with scr (n = 27) and KD1 (n = 26). See STAR Methods for calculation. ***p < 0.001.
    Figure Legend Snippet: Figure 2. Downregulation of VAMP4, but not VAMP2, impairs RE exocytosis and recycling to the plasma membrane (A) Frequency of exocytosis events in neurons transfected with TfR-SEP and TeNT-LC E234Q (n = 6) or TeNT-LC (n = 10). (B) Images of neurons co-transfected with VAMP2-SEP and TeNT-LC E234Q or TeNT-LC. VAMP2-SEP is enriched in the axon (cyan arrows) in the first case but not the second case. Scale bar, 10 mm. (C) Immunofluorescence images of endogenous VAMP4 in cells expressing GFP and a combination of shRNA targeted against VAMP4 for four days. In cells expressing GFP and the shRNA (cyan arrows), the labeling is strongly decreased compared to untransfected cells or cells expressing scramble (scr) shRNA. In cells co-expressing TfR, VAMP4-HA, and KD1, the VAMP4 staining is strong. Scale bar, 10 mm. Bottom, quantification of VAMP4 staining in the area delimited by the GFP mask (soma and dendrites). The staining is decreased by 50% in all KD conditions. The number of cells is indicated above the bars for all conditions. Comparison with scr with one-way ANOVA; *p < 0.05 and ***p < 0.001. (D) Frequency of exocytosis events recorded in cells expressing TfR-SEP and shRNAs targeted to VAMP4: scr (33 cells; 3 cells have frequencies of 0.132, 0.157, and 0.119 events.mm2.min1 and are represented above the axis limit), KD1 (23 cells), KD2 (10 cells), KD1+2 (18 cells), cells expressing VAMP4-HA (8 cells), and KD1+VAMP4-HA (12 cells). *p < 0.05 one-way ANOVA. (E) Images of neurons expressing scr or KD1 shRNAs in GFP vectors, labeled with A568-Tf (50 mg/ml) for 5 min and chased with unlabeled transferrin (2 mg/ml) at 37C for the indicated times. Scale bar, 10 mm. (F) Quantification of the Alexa568 fluorescence in the GFP mask from the pulse-chase experiments described in (E). 70 to 88 cells per condition from 4 inde- pendent experiments. Error bars represent SEM; **p < 0.01. (G) Estimation of TfR-SEP surface fraction. Top, cartoons showing the fraction of fluorescent TfR-SEP. At pH 7.4, surface receptors are fluorescent, but not at pH 5.5. Receptors in acidic intracellular organelles are not fluorescent, but become fluorescent with NH4Cl. Bottom left, images of a dendrite bathed successively in solutions at pH 7.4 (images 1, 3, and 5), pH 5.5 (image 2), and pH 7.4 containing NH4Cl (image 4). For image 4, the contrast is 23 lower than in the other images. Bottom right, quantification of the TfR-SEP surface fraction for neurons transfected with scr (n = 27) and KD1 (n = 26). See STAR Methods for calculation. ***p < 0.001.

    Techniques Used: Clinical Proteomics, Membrane, Transfection, Expressing, shRNA, Labeling, Staining, Comparison, Pulse Chase

    Figure 3. TfR-SEP and VAMP4-SEP exocytosis increase after chemical LTP (A) Images of a neuron transfected with TfR-SEP before and 15 min after induction of cLTP. Cyan crosses show the location of detected exocytosis events. Scale bar, 5 mm. (B) Normalized exocytosis frequency of neurons transfected with TfR-SEP at times relative to cLTP induction (n = 16, control [ctrl]). The light blue area denotes the time of incubation with cLTP inducing medium. The increase in frequency is significant 15 min after induction (Dunnett’s multiple comparison test, p = 0.003). In the presence of APV (100 mM), the frequency does not increase (n = 12). (C) Exocytosis frequencies before and 15 min after LTP induction. Paired t test p = 0.0008 (ctrl) and p = 0.14 (APV). (D) Normalized change in fluorescence intensity of TfR-SEP before and after cLTP induction. The increase is significant after 10 min or more (Dunnett’s multiple comparison). (E) Changes in TfR-SEP fluorescence 20 min after cLTP induction, in ctrl (carmin dots) or with APV (gray dots). Paired t test p = 0.0002 (ctrl) and p = 0.89 (APV). (F–J) Same as (A)–(E) for neurons transfected with VAMP2-SEP (n = 9) and with APV (n = 7). The increase in frequency is significant 10 min or more after induction (p = 0.002). (K–O) Same as (A)–(E) for neurons transfected with VAMP4-SEP (n = 15) and with APV (n = 15). The increase in frequency is significant 15 min or more after induction (p = 0.0082).
    Figure Legend Snippet: Figure 3. TfR-SEP and VAMP4-SEP exocytosis increase after chemical LTP (A) Images of a neuron transfected with TfR-SEP before and 15 min after induction of cLTP. Cyan crosses show the location of detected exocytosis events. Scale bar, 5 mm. (B) Normalized exocytosis frequency of neurons transfected with TfR-SEP at times relative to cLTP induction (n = 16, control [ctrl]). The light blue area denotes the time of incubation with cLTP inducing medium. The increase in frequency is significant 15 min after induction (Dunnett’s multiple comparison test, p = 0.003). In the presence of APV (100 mM), the frequency does not increase (n = 12). (C) Exocytosis frequencies before and 15 min after LTP induction. Paired t test p = 0.0008 (ctrl) and p = 0.14 (APV). (D) Normalized change in fluorescence intensity of TfR-SEP before and after cLTP induction. The increase is significant after 10 min or more (Dunnett’s multiple comparison). (E) Changes in TfR-SEP fluorescence 20 min after cLTP induction, in ctrl (carmin dots) or with APV (gray dots). Paired t test p = 0.0002 (ctrl) and p = 0.89 (APV). (F–J) Same as (A)–(E) for neurons transfected with VAMP2-SEP (n = 9) and with APV (n = 7). The increase in frequency is significant 10 min or more after induction (p = 0.002). (K–O) Same as (A)–(E) for neurons transfected with VAMP4-SEP (n = 15) and with APV (n = 15). The increase in frequency is significant 15 min or more after induction (p = 0.0082).

    Techniques Used: Transfection, Control, Incubation, Comparison

    Figure 4. Effect of TeNT-LC and VAMP4 KD on TfR-SEP exocytosis after cLTP (A) Exocytosis frequencies before and after LTP induction in neurons expressing TfR-SEP and either TeNT-LC E234Q (n = 13) or TeNT-LC (n = 13). In both conditions the increase in frequency is significant. (B) Images of dendrites before and after induction of cLTP. Scale bar, 5 mm. (C) TfR-SEP fluorescence in dendrites of neurons before and after cLTP induction. (D–F) Same as (A)–(C) for neurons expressing TfR-SEP and either scr (n = 10) or VAMP4 KD1 (n = 8) shRNA.
    Figure Legend Snippet: Figure 4. Effect of TeNT-LC and VAMP4 KD on TfR-SEP exocytosis after cLTP (A) Exocytosis frequencies before and after LTP induction in neurons expressing TfR-SEP and either TeNT-LC E234Q (n = 13) or TeNT-LC (n = 13). In both conditions the increase in frequency is significant. (B) Images of dendrites before and after induction of cLTP. Scale bar, 5 mm. (C) TfR-SEP fluorescence in dendrites of neurons before and after cLTP induction. (D–F) Same as (A)–(C) for neurons expressing TfR-SEP and either scr (n = 10) or VAMP4 KD1 (n = 8) shRNA.

    Techniques Used: Expressing, shRNA

    Figure 6. Effect of post-synaptic VAMP4 KD and TeNT on glutamatergic synaptic transmission (A) Confocal image of an organotypic hippocampal slice culture infected with scr-mScarlet lentivirus at 1 DIV and fixed at 9 DIV. Many pyramidal neurons in CA1 are brightly fluorescent. (B) DIC image of two pyramidal neurons recorded simultaneously with patch pipettes (asterisks). Epifluorescent illumination shows that the neuron on the left is brightly fluorescent (infected) while the one on the right is not (uninfected control). (C) Averages of 30 EPSCs evoked by the same stimulation in pairs of neurons, uninfected and infected with scr-mScarlet (top), shRNA KD1-mScarlet (middle), or shRNA KD2-mScarlet (botttom). Both neurons were held at 70 mV then at +40 mV. Right, plots of peak EPSC amplitude at 70 mV for each pair of neurons. In the scr condition, dots are spread around the diagonal, while in the KD1 and KD2 conditions the amplitudes are systematically higher for infected neurons. (D) Same as (C) for neurons co-electroporated with TeNT-LC and GFP. In the neurons expressing TeNT-LC, the amplitude is sytematically smaller than in control neurons.
    Figure Legend Snippet: Figure 6. Effect of post-synaptic VAMP4 KD and TeNT on glutamatergic synaptic transmission (A) Confocal image of an organotypic hippocampal slice culture infected with scr-mScarlet lentivirus at 1 DIV and fixed at 9 DIV. Many pyramidal neurons in CA1 are brightly fluorescent. (B) DIC image of two pyramidal neurons recorded simultaneously with patch pipettes (asterisks). Epifluorescent illumination shows that the neuron on the left is brightly fluorescent (infected) while the one on the right is not (uninfected control). (C) Averages of 30 EPSCs evoked by the same stimulation in pairs of neurons, uninfected and infected with scr-mScarlet (top), shRNA KD1-mScarlet (middle), or shRNA KD2-mScarlet (botttom). Both neurons were held at 70 mV then at +40 mV. Right, plots of peak EPSC amplitude at 70 mV for each pair of neurons. In the scr condition, dots are spread around the diagonal, while in the KD1 and KD2 conditions the amplitudes are systematically higher for infected neurons. (D) Same as (C) for neurons co-electroporated with TeNT-LC and GFP. In the neurons expressing TeNT-LC, the amplitude is sytematically smaller than in control neurons.

    Techniques Used: Transmission Assay, Infection, Control, shRNA, Expressing

    Figure 7. Effect of post-synaptic VAMP4 KD and TeNT on LTP (A) Average EPSCs before (black traces) and 20 to 30 min after induction of LTP (color traces) in neurons electroporated with GFP and TeNT-LC (purple) or not (gray). The dotted line shows the peak EPSC before LTP induction. Scale bars, 40 pA and 20 ms. (B) Peak EPSC amplitude normalized to baseline for pairs of neurons transfected with TeNT-LC (purple) or not (gray) (C) Ratio of EPSC amplitude 20 to 30 min after LTP induction to baseline. (D) Same as (A) for neurons transduced with lentivirus expressing scr-mScarlet (blue), shRNA KD1-mScarlet (red), and shRNA KD2-mScarlet (green). Scale bars, 40 pA and 20 ms. (E) Peak EPSC amplitude normalized to baseline for of neurons expressing the corresponding shRNAs. (F) Same as (C) for transduced neurons. ****p < 0.0001. (G) Model of dendritic TfR and AMPAR receptor trafficking.
    Figure Legend Snippet: Figure 7. Effect of post-synaptic VAMP4 KD and TeNT on LTP (A) Average EPSCs before (black traces) and 20 to 30 min after induction of LTP (color traces) in neurons electroporated with GFP and TeNT-LC (purple) or not (gray). The dotted line shows the peak EPSC before LTP induction. Scale bars, 40 pA and 20 ms. (B) Peak EPSC amplitude normalized to baseline for pairs of neurons transfected with TeNT-LC (purple) or not (gray) (C) Ratio of EPSC amplitude 20 to 30 min after LTP induction to baseline. (D) Same as (A) for neurons transduced with lentivirus expressing scr-mScarlet (blue), shRNA KD1-mScarlet (red), and shRNA KD2-mScarlet (green). Scale bars, 40 pA and 20 ms. (E) Peak EPSC amplitude normalized to baseline for of neurons expressing the corresponding shRNAs. (F) Same as (C) for transduced neurons. ****p < 0.0001. (G) Model of dendritic TfR and AMPAR receptor trafficking.

    Techniques Used: Transfection, Transduction, Expressing, shRNA



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    paper addgene 174407 vamp4 kd2 mscarlet  (Addgene inc)
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    Addgene inc paper addgene 174407 vamp4 kd2 mscarlet
    Figure 1. VAMP2 and <t>VAMP4</t> are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)
    Paper Addgene 174407 Vamp4 Kd2 Mscarlet, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/paper addgene 174407 vamp4 kd2 mscarlet/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    paper addgene 174407 vamp4 kd2 mscarlet - by Bioz Stars, 2026-03
    93/100 stars
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    Figure 1. VAMP2 and VAMP4 are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 1. VAMP2 and VAMP4 are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Transfection

    Figure 2. Downregulation of VAMP4, but not VAMP2, impairs RE exocytosis and recycling to the plasma membrane (A) Frequency of exocytosis events in neurons transfected with TfR-SEP and TeNT-LC E234Q (n = 6) or TeNT-LC (n = 10). (B) Images of neurons co-transfected with VAMP2-SEP and TeNT-LC E234Q or TeNT-LC. VAMP2-SEP is enriched in the axon (cyan arrows) in the first case but not the second case. Scale bar, 10 mm. (C) Immunofluorescence images of endogenous VAMP4 in cells expressing GFP and a combination of shRNA targeted against VAMP4 for four days. In cells expressing GFP and the shRNA (cyan arrows), the labeling is strongly decreased compared to untransfected cells or cells expressing scramble (scr) shRNA. In cells co-expressing TfR, VAMP4-HA, and KD1, the VAMP4 staining is strong. Scale bar, 10 mm. Bottom, quantification of VAMP4 staining in the area delimited by the GFP mask (soma and dendrites). The staining is decreased by 50% in all KD conditions. The number of cells is indicated above the bars for all conditions. Comparison with scr with one-way ANOVA; *p < 0.05 and ***p < 0.001. (D) Frequency of exocytosis events recorded in cells expressing TfR-SEP and shRNAs targeted to VAMP4: scr (33 cells; 3 cells have frequencies of 0.132, 0.157, and 0.119 events.mm2.min1 and are represented above the axis limit), KD1 (23 cells), KD2 (10 cells), KD1+2 (18 cells), cells expressing VAMP4-HA (8 cells), and KD1+VAMP4-HA (12 cells). *p < 0.05 one-way ANOVA. (E) Images of neurons expressing scr or KD1 shRNAs in GFP vectors, labeled with A568-Tf (50 mg/ml) for 5 min and chased with unlabeled transferrin (2 mg/ml) at 37C for the indicated times. Scale bar, 10 mm. (F) Quantification of the Alexa568 fluorescence in the GFP mask from the pulse-chase experiments described in (E). 70 to 88 cells per condition from 4 inde- pendent experiments. Error bars represent SEM; **p < 0.01. (G) Estimation of TfR-SEP surface fraction. Top, cartoons showing the fraction of fluorescent TfR-SEP. At pH 7.4, surface receptors are fluorescent, but not at pH 5.5. Receptors in acidic intracellular organelles are not fluorescent, but become fluorescent with NH4Cl. Bottom left, images of a dendrite bathed successively in solutions at pH 7.4 (images 1, 3, and 5), pH 5.5 (image 2), and pH 7.4 containing NH4Cl (image 4). For image 4, the contrast is 23 lower than in the other images. Bottom right, quantification of the TfR-SEP surface fraction for neurons transfected with scr (n = 27) and KD1 (n = 26). See STAR Methods for calculation. ***p < 0.001.

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 2. Downregulation of VAMP4, but not VAMP2, impairs RE exocytosis and recycling to the plasma membrane (A) Frequency of exocytosis events in neurons transfected with TfR-SEP and TeNT-LC E234Q (n = 6) or TeNT-LC (n = 10). (B) Images of neurons co-transfected with VAMP2-SEP and TeNT-LC E234Q or TeNT-LC. VAMP2-SEP is enriched in the axon (cyan arrows) in the first case but not the second case. Scale bar, 10 mm. (C) Immunofluorescence images of endogenous VAMP4 in cells expressing GFP and a combination of shRNA targeted against VAMP4 for four days. In cells expressing GFP and the shRNA (cyan arrows), the labeling is strongly decreased compared to untransfected cells or cells expressing scramble (scr) shRNA. In cells co-expressing TfR, VAMP4-HA, and KD1, the VAMP4 staining is strong. Scale bar, 10 mm. Bottom, quantification of VAMP4 staining in the area delimited by the GFP mask (soma and dendrites). The staining is decreased by 50% in all KD conditions. The number of cells is indicated above the bars for all conditions. Comparison with scr with one-way ANOVA; *p < 0.05 and ***p < 0.001. (D) Frequency of exocytosis events recorded in cells expressing TfR-SEP and shRNAs targeted to VAMP4: scr (33 cells; 3 cells have frequencies of 0.132, 0.157, and 0.119 events.mm2.min1 and are represented above the axis limit), KD1 (23 cells), KD2 (10 cells), KD1+2 (18 cells), cells expressing VAMP4-HA (8 cells), and KD1+VAMP4-HA (12 cells). *p < 0.05 one-way ANOVA. (E) Images of neurons expressing scr or KD1 shRNAs in GFP vectors, labeled with A568-Tf (50 mg/ml) for 5 min and chased with unlabeled transferrin (2 mg/ml) at 37C for the indicated times. Scale bar, 10 mm. (F) Quantification of the Alexa568 fluorescence in the GFP mask from the pulse-chase experiments described in (E). 70 to 88 cells per condition from 4 inde- pendent experiments. Error bars represent SEM; **p < 0.01. (G) Estimation of TfR-SEP surface fraction. Top, cartoons showing the fraction of fluorescent TfR-SEP. At pH 7.4, surface receptors are fluorescent, but not at pH 5.5. Receptors in acidic intracellular organelles are not fluorescent, but become fluorescent with NH4Cl. Bottom left, images of a dendrite bathed successively in solutions at pH 7.4 (images 1, 3, and 5), pH 5.5 (image 2), and pH 7.4 containing NH4Cl (image 4). For image 4, the contrast is 23 lower than in the other images. Bottom right, quantification of the TfR-SEP surface fraction for neurons transfected with scr (n = 27) and KD1 (n = 26). See STAR Methods for calculation. ***p < 0.001.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, shRNA, Labeling, Staining, Comparison, Pulse Chase

    Figure 3. TfR-SEP and VAMP4-SEP exocytosis increase after chemical LTP (A) Images of a neuron transfected with TfR-SEP before and 15 min after induction of cLTP. Cyan crosses show the location of detected exocytosis events. Scale bar, 5 mm. (B) Normalized exocytosis frequency of neurons transfected with TfR-SEP at times relative to cLTP induction (n = 16, control [ctrl]). The light blue area denotes the time of incubation with cLTP inducing medium. The increase in frequency is significant 15 min after induction (Dunnett’s multiple comparison test, p = 0.003). In the presence of APV (100 mM), the frequency does not increase (n = 12). (C) Exocytosis frequencies before and 15 min after LTP induction. Paired t test p = 0.0008 (ctrl) and p = 0.14 (APV). (D) Normalized change in fluorescence intensity of TfR-SEP before and after cLTP induction. The increase is significant after 10 min or more (Dunnett’s multiple comparison). (E) Changes in TfR-SEP fluorescence 20 min after cLTP induction, in ctrl (carmin dots) or with APV (gray dots). Paired t test p = 0.0002 (ctrl) and p = 0.89 (APV). (F–J) Same as (A)–(E) for neurons transfected with VAMP2-SEP (n = 9) and with APV (n = 7). The increase in frequency is significant 10 min or more after induction (p = 0.002). (K–O) Same as (A)–(E) for neurons transfected with VAMP4-SEP (n = 15) and with APV (n = 15). The increase in frequency is significant 15 min or more after induction (p = 0.0082).

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 3. TfR-SEP and VAMP4-SEP exocytosis increase after chemical LTP (A) Images of a neuron transfected with TfR-SEP before and 15 min after induction of cLTP. Cyan crosses show the location of detected exocytosis events. Scale bar, 5 mm. (B) Normalized exocytosis frequency of neurons transfected with TfR-SEP at times relative to cLTP induction (n = 16, control [ctrl]). The light blue area denotes the time of incubation with cLTP inducing medium. The increase in frequency is significant 15 min after induction (Dunnett’s multiple comparison test, p = 0.003). In the presence of APV (100 mM), the frequency does not increase (n = 12). (C) Exocytosis frequencies before and 15 min after LTP induction. Paired t test p = 0.0008 (ctrl) and p = 0.14 (APV). (D) Normalized change in fluorescence intensity of TfR-SEP before and after cLTP induction. The increase is significant after 10 min or more (Dunnett’s multiple comparison). (E) Changes in TfR-SEP fluorescence 20 min after cLTP induction, in ctrl (carmin dots) or with APV (gray dots). Paired t test p = 0.0002 (ctrl) and p = 0.89 (APV). (F–J) Same as (A)–(E) for neurons transfected with VAMP2-SEP (n = 9) and with APV (n = 7). The increase in frequency is significant 10 min or more after induction (p = 0.002). (K–O) Same as (A)–(E) for neurons transfected with VAMP4-SEP (n = 15) and with APV (n = 15). The increase in frequency is significant 15 min or more after induction (p = 0.0082).

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Transfection, Control, Incubation, Comparison

    Figure 4. Effect of TeNT-LC and VAMP4 KD on TfR-SEP exocytosis after cLTP (A) Exocytosis frequencies before and after LTP induction in neurons expressing TfR-SEP and either TeNT-LC E234Q (n = 13) or TeNT-LC (n = 13). In both conditions the increase in frequency is significant. (B) Images of dendrites before and after induction of cLTP. Scale bar, 5 mm. (C) TfR-SEP fluorescence in dendrites of neurons before and after cLTP induction. (D–F) Same as (A)–(C) for neurons expressing TfR-SEP and either scr (n = 10) or VAMP4 KD1 (n = 8) shRNA.

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 4. Effect of TeNT-LC and VAMP4 KD on TfR-SEP exocytosis after cLTP (A) Exocytosis frequencies before and after LTP induction in neurons expressing TfR-SEP and either TeNT-LC E234Q (n = 13) or TeNT-LC (n = 13). In both conditions the increase in frequency is significant. (B) Images of dendrites before and after induction of cLTP. Scale bar, 5 mm. (C) TfR-SEP fluorescence in dendrites of neurons before and after cLTP induction. (D–F) Same as (A)–(C) for neurons expressing TfR-SEP and either scr (n = 10) or VAMP4 KD1 (n = 8) shRNA.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Expressing, shRNA

    Figure 6. Effect of post-synaptic VAMP4 KD and TeNT on glutamatergic synaptic transmission (A) Confocal image of an organotypic hippocampal slice culture infected with scr-mScarlet lentivirus at 1 DIV and fixed at 9 DIV. Many pyramidal neurons in CA1 are brightly fluorescent. (B) DIC image of two pyramidal neurons recorded simultaneously with patch pipettes (asterisks). Epifluorescent illumination shows that the neuron on the left is brightly fluorescent (infected) while the one on the right is not (uninfected control). (C) Averages of 30 EPSCs evoked by the same stimulation in pairs of neurons, uninfected and infected with scr-mScarlet (top), shRNA KD1-mScarlet (middle), or shRNA KD2-mScarlet (botttom). Both neurons were held at 70 mV then at +40 mV. Right, plots of peak EPSC amplitude at 70 mV for each pair of neurons. In the scr condition, dots are spread around the diagonal, while in the KD1 and KD2 conditions the amplitudes are systematically higher for infected neurons. (D) Same as (C) for neurons co-electroporated with TeNT-LC and GFP. In the neurons expressing TeNT-LC, the amplitude is sytematically smaller than in control neurons.

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 6. Effect of post-synaptic VAMP4 KD and TeNT on glutamatergic synaptic transmission (A) Confocal image of an organotypic hippocampal slice culture infected with scr-mScarlet lentivirus at 1 DIV and fixed at 9 DIV. Many pyramidal neurons in CA1 are brightly fluorescent. (B) DIC image of two pyramidal neurons recorded simultaneously with patch pipettes (asterisks). Epifluorescent illumination shows that the neuron on the left is brightly fluorescent (infected) while the one on the right is not (uninfected control). (C) Averages of 30 EPSCs evoked by the same stimulation in pairs of neurons, uninfected and infected with scr-mScarlet (top), shRNA KD1-mScarlet (middle), or shRNA KD2-mScarlet (botttom). Both neurons were held at 70 mV then at +40 mV. Right, plots of peak EPSC amplitude at 70 mV for each pair of neurons. In the scr condition, dots are spread around the diagonal, while in the KD1 and KD2 conditions the amplitudes are systematically higher for infected neurons. (D) Same as (C) for neurons co-electroporated with TeNT-LC and GFP. In the neurons expressing TeNT-LC, the amplitude is sytematically smaller than in control neurons.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Transmission Assay, Infection, Control, shRNA, Expressing

    Figure 7. Effect of post-synaptic VAMP4 KD and TeNT on LTP (A) Average EPSCs before (black traces) and 20 to 30 min after induction of LTP (color traces) in neurons electroporated with GFP and TeNT-LC (purple) or not (gray). The dotted line shows the peak EPSC before LTP induction. Scale bars, 40 pA and 20 ms. (B) Peak EPSC amplitude normalized to baseline for pairs of neurons transfected with TeNT-LC (purple) or not (gray) (C) Ratio of EPSC amplitude 20 to 30 min after LTP induction to baseline. (D) Same as (A) for neurons transduced with lentivirus expressing scr-mScarlet (blue), shRNA KD1-mScarlet (red), and shRNA KD2-mScarlet (green). Scale bars, 40 pA and 20 ms. (E) Peak EPSC amplitude normalized to baseline for of neurons expressing the corresponding shRNAs. (F) Same as (C) for transduced neurons. ****p < 0.0001. (G) Model of dendritic TfR and AMPAR receptor trafficking.

    Journal: Cell reports

    Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

    doi: 10.1016/j.celrep.2021.109678

    Figure Lengend Snippet: Figure 7. Effect of post-synaptic VAMP4 KD and TeNT on LTP (A) Average EPSCs before (black traces) and 20 to 30 min after induction of LTP (color traces) in neurons electroporated with GFP and TeNT-LC (purple) or not (gray). The dotted line shows the peak EPSC before LTP induction. Scale bars, 40 pA and 20 ms. (B) Peak EPSC amplitude normalized to baseline for pairs of neurons transfected with TeNT-LC (purple) or not (gray) (C) Ratio of EPSC amplitude 20 to 30 min after LTP induction to baseline. (D) Same as (A) for neurons transduced with lentivirus expressing scr-mScarlet (blue), shRNA KD1-mScarlet (red), and shRNA KD2-mScarlet (green). Scale bars, 40 pA and 20 ms. (E) Peak EPSC amplitude normalized to baseline for of neurons expressing the corresponding shRNAs. (F) Same as (C) for transduced neurons. ****p < 0.0001. (G) Model of dendritic TfR and AMPAR receptor trafficking.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

    Techniques: Transfection, Transduction, Expressing, shRNA