polyclonal antibody to syt1  (Cusabio)

Journal: Frontiers in Behavioral Neuroscience

Article Title: Altered cognition and anxiety in adolescent offspring whose mothers underwent different-pattern maternal sleep deprivation, and cognition link to hippocampal expressions of Bdnf and Syt-1

doi: 10.3389/fnbeh.2022.1066725

Figure Lengend Snippet: Effects of MSD on the expression of hippocampal of BDNF and Syt-1 proteins in the offspring. (A) The expression levels of BDNF ( n = 12, 6 females and 6 males in each treatment group) and Syt-1 ( n = 12, 6 females and 6 males in each treatment group) proteins in the hippocampus at 2 months. The results of protein quantification are depicted in (B,C) . ** P < 0.01 vs. Male CON group; ## P < 0.01 vs. Male CSD3h group; $$ P < 0.01 vs. Male CSD6h group; aa P < 0.01 vs. Female CON group; bb P < 0.01 vs. Female CSD3h group; cc P < 0.01 vs. Female CSD6h group.

Article Snippet: The membrane was subsequently incubated with the rabbit anti-BDNF (1:1000; Abcam, Cambridge, UK) and rabbit anti-Syt-1 (1:1000; Bioss, Beijing, China) primary antibodies, and the horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (Zsbio, ZB-2301) secondary antibody, according to the manufacturer's instructions.

Techniques: Expressing

Journal: Frontiers in Behavioral Neuroscience

Article Title: Altered cognition and anxiety in adolescent offspring whose mothers underwent different-pattern maternal sleep deprivation, and cognition link to hippocampal expressions of Bdnf and Syt-1

doi: 10.3389/fnbeh.2022.1066725

Figure Lengend Snippet: The effects of MSD on the expression of hippocampal of BDNF and Syt-1 mRNA in the offspring. The mRNA expression levels of BDNF ( n = 16, 8 females and 8 males in each treatment group) and Syt1 ( n = 16, 8 females and 8 males in each treatment group) in the hippocampus at 2 months. The results of mRNA quantification are depicted in (A,B) . ** P < 0.01 vs. Male CON group; # P < 0.05 and ## P < 0.01 vs. Male CSD3h group; $ P < 0.05 and $$ P < 0.01 vs. Male CSD6h group; a P < 0.05 and aa P < 0.01 vs. Female CON group; b P < 0.05 and bb P < 0.01 vs. Female CSD3h group; c P < 0.05 and cc P < 0.01 vs. Female CSD6h group.

Article Snippet: The membrane was subsequently incubated with the rabbit anti-BDNF (1:1000; Abcam, Cambridge, UK) and rabbit anti-Syt-1 (1:1000; Bioss, Beijing, China) primary antibodies, and the horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (Zsbio, ZB-2301) secondary antibody, according to the manufacturer's instructions.

Techniques: Expressing

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Virus, Recombinant, cDNA Synthesis, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal antibody anti-Syt1 , Synaptic Systems , Cat#105103; RRID: AB_11042457.

Techniques: Virus, Recombinant, cDNA Synthesis, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Antibodies used were rabbit polyclonal antibody (RpAb) to Syt1 (1:1000) from Synaptic Systems (105103), mouse monoclonal antibody (MmAb) to Syt2 (1:1000) from Abnova (clone ZPN-1), RpAb to Cre (1:1000) from Cell Signaling (15036S), MmAb to α-tubulin (1:1000) from Sigma-Aldrich (clone DM1A), RpAb to ARSI (1:1000) from Thermo Fisher Scientific (PA5-49984), and RpAb to SLC6Α7 (1:500) from almone labs (AGT-013).

Techniques: Recombinant, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software

Journal: Molecular & cellular proteomics : MCP

Article Title: Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

doi: 10.1016/j.mcpro.2022.100222

Figure Lengend Snippet: FIG. 3. Immature neurons with low expression of Syt1 exhibit enhanced tolerance to cerebral ischemia in vivo and OGD in vitro. A and B, representative Western blot image (A) and quantitative analysis (B) of the expression level of Syt1 in cultured immature and mature

Article Snippet: The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum (16210064, Thermo Fisher(v/v)) in trisbuffered saline with 0.1% Tween 20 (93773, Sigma) at room temperature for 1 h. Monoclonal antibodies to β-actin (HC201-02, TransGen Biotech), polyclonal antibody to Syt1 (CSB-PA019553GA01HU, CUSABIO Biotech), rabbit polyclonal anti-phosphotyrosine (Cat# PTM-702, PTM BIO) were employed for Western blot analyses as primary antibodies at 4 ◦C overnight.

Techniques: Expressing, In Vivo, In Vitro, Western Blot, Cell Culture

Journal: Molecular & cellular proteomics : MCP

Article Title: Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

doi: 10.1016/j.mcpro.2022.100222

Figure Lengend Snippet: FIG. 4. Administration of the Tat-Syt1T112A peptide protects against neuronal injury in cerebral ischemia. A, schematic diagram of mouse full-length Syt1 and tat peptides for Syt1WT and Syt1T112A. B, representative images of hippocampal neurons incubated with either Tat-V5

Article Snippet: The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum (16210064, Thermo Fisher(v/v)) in trisbuffered saline with 0.1% Tween 20 (93773, Sigma) at room temperature for 1 h. Monoclonal antibodies to β-actin (HC201-02, TransGen Biotech), polyclonal antibody to Syt1 (CSB-PA019553GA01HU, CUSABIO Biotech), rabbit polyclonal anti-phosphotyrosine (Cat# PTM-702, PTM BIO) were employed for Western blot analyses as primary antibodies at 4 ◦C overnight.

Techniques: Incubation

Journal: Molecular & cellular proteomics : MCP

Article Title: Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

doi: 10.1016/j.mcpro.2022.100222

Figure Lengend Snippet: FIG. 5. Kcnq2 interacts with Syt1 and alleviates Syt1-mediated neuronal injury by OGD treatment. A and B, representative LC–MS/MS spectrum of Kcnq2 phosphorylation at the S52 site (A) and quantification of KCNQ phosphorylation at the S52 site and Kcnq2 protein expression level (B) in the hippocampus after sham or acute MCAO ischemia for 2 h. (sham: n = 3 repeat MS, MCAO: n = 3 repeat MS). C and D, representative images (C) and quantitative analysis (D) of the average length of dendrites of cultured hippocampal neurons transfected with pFUGW-GFP and either an empty vector or a plasmid encoding Kcnq2WT, Kcnq2S52A, or Kcnq2S52D at DIV 10 followed by control or OGD treatment for 2 h at DIV 14. The scale bar represents 100 μm and 10 μm (magnified images). Transfected neurons were chosen randomly, and images were acquired using a confocal microscope. (control: vector: n = 35 cells/3 cultures, Kcnq2WT: n = 34 cells/3 cultures, Kcnq2S52A: n =

Article Snippet: The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum (16210064, Thermo Fisher(v/v)) in trisbuffered saline with 0.1% Tween 20 (93773, Sigma) at room temperature for 1 h. Monoclonal antibodies to β-actin (HC201-02, TransGen Biotech), polyclonal antibody to Syt1 (CSB-PA019553GA01HU, CUSABIO Biotech), rabbit polyclonal anti-phosphotyrosine (Cat# PTM-702, PTM BIO) were employed for Western blot analyses as primary antibodies at 4 ◦C overnight.

Techniques: Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Expressing, Cell Culture, Transfection, Plasmid Preparation, Control, Microscopy

Journal: Molecular & cellular proteomics : MCP

Article Title: Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

doi: 10.1016/j.mcpro.2022.100222

Figure Lengend Snippet: FIG. 6. Kcnq2 interacts with Anxa6 and alleviates neuronal injury by inhibiting Syt1. A, representative image of the glutathione-S- transferase (GST) pull-down assay of Flag-tagged Kcnq2 (Kcnq2-Flag) to immobilize GST fused to full-length Anxa6 or GST alone followed

Article Snippet: The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum (16210064, Thermo Fisher(v/v)) in trisbuffered saline with 0.1% Tween 20 (93773, Sigma) at room temperature for 1 h. Monoclonal antibodies to β-actin (HC201-02, TransGen Biotech), polyclonal antibody to Syt1 (CSB-PA019553GA01HU, CUSABIO Biotech), rabbit polyclonal anti-phosphotyrosine (Cat# PTM-702, PTM BIO) were employed for Western blot analyses as primary antibodies at 4 ◦C overnight.

Techniques: Pull Down Assay

Journal: Nature Communications

Article Title: Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R

doi: 10.1038/s41467-021-27462-7

Figure Lengend Snippet: a Neurons were treated with sulfo-NHS-S-S-biotin to label cell-surface proteins. Following a 6-h incubation, allowing for internalization, remaining cell-surface proteins were stripped of their labels with glutathione. After a further 18 h of incubation, allowing for recycling, the neurons were again treated with glutathione. Lysates representing surface, endocytosed or non-recycled pools, were collected on streptavidin-coupled beads, immunostained for TNR, immobilized on glass slides and imaged with confocal microscopy. b Example beads collecting TNR pools, or controls incubated without primary antibodies. Scale bar = 2 µm. c A quantification of TNR fluorescence intensity normalized to the ‘surface’ mean in the corresponding experiment, indicates that a large fraction of TNR molecules endocytose within 6 h, and many subsequently resurface within 24 h. As positive/negative controls, the synaptic vesicle protein Syt1, well-known to recycle, and the intracellular protein calmodulin were tested. The plots are scaled by the ratio between the ‘surface’ mean for these proteins and that of TNR. N = 4 (TNR) and 3 (Syt1/calmodulin) independent experiments with >100 (TNR) and >50 (Syt/calmodulin) beads. d , e As additional controls, the lysosomal marker LAMP1, known to endocytose but scarcely recycle, and myelin basic protein (MBP), which should not endocytose, were also tested. Scale bar = 1 μm. e Quantification of LAMP1/MBP fluorescence intensity, normalized to the ‘surface’ mean of the corresponding experiment. N = 3 independent experiments with > 50 beads. Statistical significance was evaluated using repeated-measures one-way ANOVA, ( c TNR: F 1.153,3.458 = 28.29, ** p = 0.009; Syt1: F 1.007,2.014 = 62.98, * p = 0.015; calmodulin: F 1,2 = 0.016, p = 0.912; e LAMP1: F 1.293,2.585 = 19.6, * p = 0.028; MBP: F 1.52,3.041 = 28337, *** p < 0.001), followed by the Holm-Sidak multiple comparisons test comparing ‘surface’/‘endocytosed’ and ‘endocytosed’/‘non-recycled’ ( c TNR: * p = 0.032, * p = 0.021; Syt1: *p = 0.044, * p = 0.044; calmodulin: p = 0.933, p = 0.993; e LAMP1: *p = 0.045, p = 0.162; MBP: *** p < 0.001, p = 0.068). Data represent the mean ± SEM, dots indicate individual experiments. Source data are provided in Source Data file.

Article Snippet: For the analysis of stimulus-induced vesicle release in cultured neurons, the aggregates were added for 30 min, after which the neurons were incubated for 5 min with 1:100 polyclonal rabbit antibodies directed against the lumenal domain of Syt1 conjugated to the fluorescent dye Oyster488 (#105 103C2; Synaptic Systems, Göttingen, Germany) in either plain Tyrode (stimulated condition) or Ca 2+ -free Tyrode containing 1 mM EGTA and 1 μM TTX (#1069; Tocris Bioscience, Germany) (unstimulated condition).

Techniques: Incubation, Confocal Microscopy, Fluorescence, Marker

Journal: Nature Communications

Article Title: Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R

doi: 10.1038/s41467-021-27462-7

Figure Lengend Snippet: a The TNR epitopes in the ECM were blocked as in the previous experiments, and 12 h later the cultures were incubated with fluorophore-conjugated TNR antibodies (magenta) and with fluorophore-conjugated antibodies for the intra-vesicular domain of Syt1 (green), which reveal the synaptic vesicle pool that undergoes exo- and endocytosis (the active pool). The size of this pool is a measure of the activity of the respective boutons. The panels show example synapses with different active vesicle pools, imaged in STED (TNR) and confocal (Syt1). Scale bar = 300 nm. The graph shows the mean fluorescence intensities normalized to the median intensity of the respective experiment. The Syt1 intensities are binned to include an equal number of synapse images. An analysis of the correlation of the TNR signal at Syt1-labeled synapses indicates that the TNR signals correlate strongly with the size of the active vesicle pool in the respective boutons. N = 3 independent experiments, with >1100 synapses per datapoint, Spearman’s ⍴ = 0.927, ** *p = 6.489 × 10 −7 (two-sided). b Newly-emerged TNR epitopes (magenta) were labeled after 12 h as in panel a, and the neuronal plasma membrane was visualized with DiO (green). The panels show example spines with different sizes, imaged in STED (TNR) and confocal (DiO). Scale bar = 300 nm. The graph shows the mean fluorescence intensity of TNR and the mean synapse area, normalized to the median values in the respective experiment. The synapse area values are binned to include an equal number of synapse images. An analysis of the correlation of the TNR signal at DiO-labeled spines indicates that the TNR signals correlate strongly with the size of the dendritic spine for newly-emerged TNR epitopes. N = 3 independent experiments, with >280 synapses per datapoint, Spearman’s ⍴ = 0.862, ** *p = 3.601 × 10 −5 (two-sided). All data represent the mean ± SEM, with dots indicating individual experiments. Source data are provided in Source Data file.

Article Snippet: For the analysis of stimulus-induced vesicle release in cultured neurons, the aggregates were added for 30 min, after which the neurons were incubated for 5 min with 1:100 polyclonal rabbit antibodies directed against the lumenal domain of Syt1 conjugated to the fluorescent dye Oyster488 (#105 103C2; Synaptic Systems, Göttingen, Germany) in either plain Tyrode (stimulated condition) or Ca 2+ -free Tyrode containing 1 mM EGTA and 1 μM TTX (#1069; Tocris Bioscience, Germany) (unstimulated condition).

Techniques: Incubation, Activity Assay, Fluorescence, Labeling, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R

doi: 10.1038/s41467-021-27462-7

Figure Lengend Snippet: a Assay to perturb TNR recycling: newly-emerged TNR epitopes were labeled 12 h post-blocking with biotinylated antibodies, and bound to large aggregates of antibodies. As control, all other epitopes (non-recycling) were labeled. b STED images of aggregates. Scale bar = 1 µm. c Histogram of aggregate size (FWHM). N = 4 independent experiments, 995 aggregates. d Neurons were incubated with aggregates for 30 min. Synaptic activity was assessed by uptake of Syt1 antibodies (as in Fig. ). Without stimulation, Syt1 antibodies detect the surface vesicle population (40–50% of actively-recycling vesicles ). Stimulation results in signal increase (exo-/endocytosis of new vesicles) in controls, but not in aggregate-treated cultures (epifluorescence). Scale bar = 4 µm. e Quantification of Syt1 fluorescence intensity confirms this observation and indicates that tagging all other epitopes has no effects. N = 4 (‘new epitopes’)/3 (‘all other’) independent experiments, ≥15 neurons per datapoint. Repeated-measures ANOVA on rank (‘new epitopes’: F 1,6 = 12.54, * p = 0.012; ‘all other epitopes’: F 1,4 = 1.5, p = 0.288) for the interaction Stim/ctrl x + /− Aggregates), followed by Sidak’s multiple comparisons test (‘new epitopes’: * p = 0.02, p = 0.419; ‘all other epitopes’: ** p = 0.002, *** p < 0.001 for ‘stim’ vs. ‘ctrl’ for untreated and treated neurons, respectively). f , g Effect of recycling perturbation on synapse structure. Dissociated cultures ( f ) and organotypic hippocampal slices ( g ) were treated with aggregates for 12 h. Plasma membranes were visualized with DiO ( f ) or by infection with AAV9-Syn-eGFP ( g ), in averaged spines or individual examples (insets). Scale bar = 300 nm ( f ), 500 nm ( g ). N = 3 independent experiments, >80 ( f ), >60 ( g ) synapses per condition. One-way ANOVA ( f : F 2, 6 = 5.269, * p = 0.05) or repeated-measures one-way ANOVA ( g : F 1.041, 2.083 = 20.76, * p = 0.042), followed by Fisher’s LSD ( f : ** p = 0.005, p = 0.418; g : *p = 0.025, p = 0.16), to compare ‘all other e p itopes’/‘new epitopes’ and ‘all other epitopes’/‘Tyrode’, respectively. Data represent mean ± SEM, dots indicate individual experiments ( d – g ). Source data are provided in Source Data file.

Article Snippet: For the analysis of stimulus-induced vesicle release in cultured neurons, the aggregates were added for 30 min, after which the neurons were incubated for 5 min with 1:100 polyclonal rabbit antibodies directed against the lumenal domain of Syt1 conjugated to the fluorescent dye Oyster488 (#105 103C2; Synaptic Systems, Göttingen, Germany) in either plain Tyrode (stimulated condition) or Ca 2+ -free Tyrode containing 1 mM EGTA and 1 μM TTX (#1069; Tocris Bioscience, Germany) (unstimulated condition).

Techniques: Labeling, Blocking Assay, Control, Incubation, Activity Assay, Fluorescence, Clinical Proteomics, Infection