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: Theranostics

Article Title: ANKRD22, a novel tumor microenvironment-induced mitochondrial protein promotes metabolic reprogramming of colorectal cancer cells

doi: 10.7150/thno.37472

Figure Lengend Snippet: Synergistic effect of ANKRD22 and E-Syt1 results in the abnormal accumulation of lipids in mitochondria of CCICs. (A) Lipid metabolism-related proteins of the pull-down products of ANKRD22-overexpressing cells cultured in conventional 2D condition or enriched by organoid culture. (B) Expression of E-Syt1 in the normal colorectal epithelium and CRC epithelium by IHC. (C) Co-IP verification of the interaction between ANKRD22 and E-Syt1 in conventional 2D-cultured or organoid-cultured RKO cells with Halo-ANKRD22 overexpression. (D-E) Co-IP verification of the interaction between ANKRD22 and truncated E-Syt1s. Vectors encoding different regions of E-Syt1 were transfected into SGC-7901 cells that stably expressed Halo-ANKRD22 and performed Co-IP according to the Halo-tag pull-down protocol; the pull-down eluent of SGC-7901 cells was used as a negative control. (F) Co-IP verification of the interaction between ANT2 and E-Syt1. Flag-SLC25A5 /pcDNA3.1(-) or pcDNA3.1(-) plasmids were transfected into E-Syt1-overexpressing 293T cells. Co-IP assay was conducted after 48 hours. (G) Influence of SLC25A5 knockdown on the interaction between ANKRD22 and E-Syt1. SLC25A5 -knockdown RKO cells and scrambled shRNA-infected RKO cells were infected with Halo-ANKRD22 lentivirus, and the pull-down assay was performed; the pull-down eluent of scrambled shRNA-infected RKO cells was used as a negative control. (H) Influence of E-Syt1 knockdown on the interaction between ANKRD22 and ANT2. ESYT1 -knockdown RKO cells and scrambled shRNA-infected RKO cells were infected with Halo-ANKRD22 lentivirus and the pull-down assay was performed; the pull-down eluent of scrambled shRNA-infected RKO cells was used as a negative control. (I-K) Effect of mitochondria-localized E-Syt1 on mitochondrial morphology. An E-Syt1 sequence fused with a mitochondrial localization sequence at the C-terminus was transfected into ANKRD22-overexpressing 293T cells and observed by electron microscopy. The percentage of vacuole-like and crista-reduced mitochondria in each cell was calculated (n=6). (L) Detection of E-Syt1 in the mitochondria of organoid-cultured RKO cells (Halo-ANKRD22 overexpression vs control) by WB. β-Tubulin and VDAC1 were internal references for the cytoplasmic (C) and mitochondrial (M) fractions, respectively. (M) Effect of ANKRD22 and E-Syt1 on cytoplasmic Ca 2+ level. Fluo-4 staining was used to detect the effect of ANKRD22 or synergistic effect of ANKRD22 and E-Syt1 on the cytoplasmic Ca 2+ level of SGC-7901 cell in vitro . Three replicates were performed for each group. The fluorescence intensity of FITC was detected by FCM. The data were analyzed by Student's t -test and are presented as mean±SD, *** p <0.001. (N) Effect of Ankrd22 knockout on cytoplasmic Ca 2+ level. Primary colorectal cells of Ankrd22 -/- mice (n=3) and wild-type C57BL/6 mice (n=3) were stained with fluo-4. The average fluorescence intensity was analyzed by Student's t -test, and data are presented as mean±SD, * p < 0.05.

Article Snippet: The following primary antibodies were used in this study: β-Tubulin (HuaBio, China), p38 MAPK (Cell Signaling Technology, USA), phos-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), phos-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology), MAX (Abcam, UK), VDAC1 (Abcam), MDH2 (Abcam), Histone H3 (Cell Signaling Technology), AMPK (Cell Signaling Technology), phos-AMPK(Thr172) (Cell Signaling Technology), phos-ACC (Ser79) (Cell Signaling Technology), PDK1 (HuaBio), ANT2 (Celling Signaling Technology), Flag Tag (HuaBio), LC3B (Cell Signaling Technology), SQSTM1/P62 (Cell Signaling Technology), E-Syt1 (Proteintech, China), DAG (LifeSpan BioSciences, USA), PIP2 (Invitrogen, USA), p53 (Cell Signaling Technology).

Techniques: Cell Culture, Expressing, Co-Immunoprecipitation Assay, Over Expression, Transfection, Stable Transfection, Negative Control, Knockdown, shRNA, Infection, Pull Down Assay, Sequencing, Electron Microscopy, Control, Staining, In Vitro, Fluorescence, Knock-Out

Journal: Journal of Virology

Article Title: Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

doi: 10.1128/jvi.01281-17

Figure Lengend Snippet: Fig. 3: E-Syt1 interacts with HSV-1gM in infected cells. HeLa cells were infected 728

Article Snippet: Primary antibodies and dilution used in this study were as follows: anti-336 myc rabbit polyclonal (1:1000; 2272, Cell Signaling Technology), anti-myc mouse 337 polyclonal ( 9B11) (1:100; 2276, Cell Signaling Technology) anti-HA mouse monoclonal 338 (1:1000; SC-7392, Santa Cruz), anti E-Syt1 rabbit polyclonal (1:1000; A303-362A, 339 Bethyl Laboratories), anti E-Syt2 rabbit polyclonal (1:1000; NBP1-59988, Novus 340 Biologicals), anti E-Syt3 rabbit polyclonal (1:1000; NBP1-91354, Novus Biologicals), 341 anti β-actin mouse monoclonal (1:2500; ab6276, Abcam), anti γ-Tubulin mouse 342 monoclonal (1:5000; T6557, Sigma-Aldrich), anti-HSV VP5 mouse monoclonal (1:2000; 343 Virusys), anti-HSV gM rabbit polyclonal PAS980 (1:1000; courtesy of Dr. Lynn 344 Enquist), anti-HSV gM rabbit polyclonal 4c10 (1:000; courtesy of Dr. Joel Baines) and 345 anti-human DDX3 rabbit R648 polyclonal (1:4000; courtesy of Dr. A. Patel).

Techniques: Infection

Journal: Journal of Virology

Article Title: Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

doi: 10.1128/jvi.01281-17

Figure Lengend Snippet: Fig. 5: Impact of the virus on E-Syt1 expression. HeLa cells were mock treated or 750

Article Snippet: Primary antibodies and dilution used in this study were as follows: anti-336 myc rabbit polyclonal (1:1000; 2272, Cell Signaling Technology), anti-myc mouse 337 polyclonal ( 9B11) (1:100; 2276, Cell Signaling Technology) anti-HA mouse monoclonal 338 (1:1000; SC-7392, Santa Cruz), anti E-Syt1 rabbit polyclonal (1:1000; A303-362A, 339 Bethyl Laboratories), anti E-Syt2 rabbit polyclonal (1:1000; NBP1-59988, Novus 340 Biologicals), anti E-Syt3 rabbit polyclonal (1:1000; NBP1-91354, Novus Biologicals), 341 anti β-actin mouse monoclonal (1:2500; ab6276, Abcam), anti γ-Tubulin mouse 342 monoclonal (1:5000; T6557, Sigma-Aldrich), anti-HSV VP5 mouse monoclonal (1:2000; 343 Virusys), anti-HSV gM rabbit polyclonal PAS980 (1:1000; courtesy of Dr. Lynn 344 Enquist), anti-HSV gM rabbit polyclonal 4c10 (1:000; courtesy of Dr. Joel Baines) and 345 anti-human DDX3 rabbit R648 polyclonal (1:4000; courtesy of Dr. A. Patel).

Techniques: Virus, Expressing

Journal: Journal of Virology

Article Title: Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

doi: 10.1128/jvi.01281-17

Figure Lengend Snippet: Fig. 9: E-Syt1 and E-Syt3 down regulation increases cell-to-cell spread. The 798

Article Snippet: Primary antibodies and dilution used in this study were as follows: anti-336 myc rabbit polyclonal (1:1000; 2272, Cell Signaling Technology), anti-myc mouse 337 polyclonal ( 9B11) (1:100; 2276, Cell Signaling Technology) anti-HA mouse monoclonal 338 (1:1000; SC-7392, Santa Cruz), anti E-Syt1 rabbit polyclonal (1:1000; A303-362A, 339 Bethyl Laboratories), anti E-Syt2 rabbit polyclonal (1:1000; NBP1-59988, Novus 340 Biologicals), anti E-Syt3 rabbit polyclonal (1:1000; NBP1-91354, Novus Biologicals), 341 anti β-actin mouse monoclonal (1:2500; ab6276, Abcam), anti γ-Tubulin mouse 342 monoclonal (1:5000; T6557, Sigma-Aldrich), anti-HSV VP5 mouse monoclonal (1:2000; 343 Virusys), anti-HSV gM rabbit polyclonal PAS980 (1:1000; courtesy of Dr. Lynn 344 Enquist), anti-HSV gM rabbit polyclonal 4c10 (1:000; courtesy of Dr. Joel Baines) and 345 anti-human DDX3 rabbit R648 polyclonal (1:4000; courtesy of Dr. A. Patel).

Techniques:

Journal: Journal of Virology

Article Title: Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

doi: 10.1128/jvi.01281-17

Figure Lengend Snippet: Fig. 11: Overexpression of E-Syt1 or E-Syt-3 reduces viral yields. HeLa cells 818

Article Snippet: Primary antibodies and dilution used in this study were as follows: anti-336 myc rabbit polyclonal (1:1000; 2272, Cell Signaling Technology), anti-myc mouse 337 polyclonal ( 9B11) (1:100; 2276, Cell Signaling Technology) anti-HA mouse monoclonal 338 (1:1000; SC-7392, Santa Cruz), anti E-Syt1 rabbit polyclonal (1:1000; A303-362A, 339 Bethyl Laboratories), anti E-Syt2 rabbit polyclonal (1:1000; NBP1-59988, Novus 340 Biologicals), anti E-Syt3 rabbit polyclonal (1:1000; NBP1-91354, Novus Biologicals), 341 anti β-actin mouse monoclonal (1:2500; ab6276, Abcam), anti γ-Tubulin mouse 342 monoclonal (1:5000; T6557, Sigma-Aldrich), anti-HSV VP5 mouse monoclonal (1:2000; 343 Virusys), anti-HSV gM rabbit polyclonal PAS980 (1:1000; courtesy of Dr. Lynn 344 Enquist), anti-HSV gM rabbit polyclonal 4c10 (1:000; courtesy of Dr. Joel Baines) and 345 anti-human DDX3 rabbit R648 polyclonal (1:4000; courtesy of Dr. A. Patel).

Techniques: Over Expression

Journal: Journal of Virology

Article Title: Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

doi: 10.1128/jvi.01281-17

Figure Lengend Snippet: Fig. 13: E-Syt1 and E-Syt3 knockdown increase cell-to-cell fusion in infected 843

Article Snippet: Primary antibodies and dilution used in this study were as follows: anti-336 myc rabbit polyclonal (1:1000; 2272, Cell Signaling Technology), anti-myc mouse 337 polyclonal ( 9B11) (1:100; 2276, Cell Signaling Technology) anti-HA mouse monoclonal 338 (1:1000; SC-7392, Santa Cruz), anti E-Syt1 rabbit polyclonal (1:1000; A303-362A, 339 Bethyl Laboratories), anti E-Syt2 rabbit polyclonal (1:1000; NBP1-59988, Novus 340 Biologicals), anti E-Syt3 rabbit polyclonal (1:1000; NBP1-91354, Novus Biologicals), 341 anti β-actin mouse monoclonal (1:2500; ab6276, Abcam), anti γ-Tubulin mouse 342 monoclonal (1:5000; T6557, Sigma-Aldrich), anti-HSV VP5 mouse monoclonal (1:2000; 343 Virusys), anti-HSV gM rabbit polyclonal PAS980 (1:1000; courtesy of Dr. Lynn 344 Enquist), anti-HSV gM rabbit polyclonal 4c10 (1:000; courtesy of Dr. Joel Baines) and 345 anti-human DDX3 rabbit R648 polyclonal (1:4000; courtesy of Dr. A. Patel).

Techniques: Knockdown, Infection

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: Phosphorylation of Syt1 at Thr112 accelerates neuronal injury during OGD. A , representative LC–MS/MS spectrum of phosphorylation of Syt1 at the threonine 112 (T112) site in the hippocampus 2 h after sham or acute MCAO ischemia. B , quantification of the phosphorylation level of Syt1 at the T112 site and the protein expression level of Syt1 2 h after ischemia. (sham: n = 3 repeat MS, MCAO: n = 3 repeat MS). C , representative images of hippocampal neurons transfected with pFUGW-GFP and either an empty vector (vector) or a plasmid encoding Syt1 WT , Syt1 T112A , or Syt1 T112D at DIV 10 and subjected to control or OGD 2 h treatment 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. D , quantitative analysis of the average length of dendrites of neurons presented in ( C ). (control: vector: n = 31 cells/3 cultures, Syt1 WT : n = 34 cells/3 cultures, Syt1 T112A : n = 37 cells/3 cultures, Syt1 T112D : n = 30 cells/3 cultures; OGD 2 h: vector: n = 38 cells/3 cultures, Syt1 WT : n = 37 cells/3 cultures, Syt1 T112A : n = 40 cells/3 cultures, Syt1 T112D : n = 38 cells/3 cultures). E and F , representative images of hippocampal neurons transfected together with pFUGW-GFP and either an empty vector (control) or a plasmid encoding Syt1 WT or Syt1 T112D at DIV 10 and subjected to control ( E ) or OGD for 2 h ( F ) treatment at DIV 14 with DMSO, CNQX, or MgCl 2 . The scale bar represents 100 μm and 10 μm (magnified images). Transfected neurons were chosen randomly, and images were acquired using a confocal microscope. G , quantitative analysis of the average length of dendrites of neurons presented in ( E ) and ( F ). (control: vector+DMSO: n = 23 cells/3 cultures, vector+CNQX: n = 22 cells/3 cultures, vector+Mgcl 2 : n = 25 cells/3 cultures, Syt1 WT +DMSO: n = 18 cells/3 cultures, Syt1 WT +CNQX: n = 20 cells/3 cultures, Syt1 WT +Mgcl 2 : n = 19 cells/3 cultures, Syt1 T112D +DMSO: n = 16 cells/3 cultures, Syt1 T112D +CNQX: n = 19 cells/3 cultures, Syt1 T112D +Mgcl 2 : n = 17 cells/3 cultures; OGD 2 h: vector+DMSO: n = 22 cells/3 cultures, vector+CNQX: n = 20 cells/3 cultures, vector+Mgcl 2 : n = 23 cells/3 cultures, Syt1 WT +DMSO: n = 23 cells/3 cultures, Syt1 WT +CNQX: n = 20 cells/3 cultures, Syt1 WT +Mgcl2: n = 24 cells/3 cultures, Syt1 T112D +DMSO: n = 23 cells/3 cultures, Syt1 T112D +CNQX: n = 20 cells/3 cultures, Syt1 T112D +Mgcl2: n = 22 cells/3 cultures). Data are presented as means ± SEM (error bars). The number of repeat times ( B ) and neurons ( D and G ) analyzed are indicated in columns. Unpaired Student’s t tests ( B ) and one-way ANOVA tests followed by Tukey’s multiple comparison tests ( D and G ) were performed. All experiments were repeated at least three times. DMSO, dimethylsulfoxide; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose deprivation.

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 tris-buffered 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: Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Transfection, Plasmid Preparation, Control, Microscopy, Comparison

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: 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 hippocampal neurons (immature neurons: n = 4 cultures, mature neurons, n = 4 cultures). C and D , representative Western blot image ( C ) and quantitative analysis ( D ) of the expression level of Syt1 in immature and mature mouse hippocampal tissue lysates (immature mice: n = 4 mice, mature mice, n = 4 mice). E , representative images of cultured mature and immature hippocampal neurons immunostained with an antibody against MAP2 after control or 2 h of OGD treatment. The scale bar represents 100 μm and 20 μm (magnified images). Stained neurons were chosen randomly, and images were acquired using a confocal microscope. F , quantitative analysis of the average length of MAP2 and branch number of neurons presented in ( E ) (Control: DIV 14: n = 36 cells/3 cultures, DIV 4: n = 36 cells/3 cultures; OGD 2 h: DIV 14: n = 36 cells/3 cultures, DIV 4: n = 36 cells/3 cultures). G , LDH release from cultured mature and immature hippocampal neurons after 2 h of control or 2 h of OGD treatment. (control: DIV 14: n = 6 wells/3 cultures, DIV 4: n = 11 wells/3 cultures; OGD 2 h: DIV 14: n = 4 wells/3 cultures, DIV 4: n = 11 wells/3 cultures). H , representative images of blood flow of mature and immature mice subjected to sham or MCAO operation for 24 h. I , quantitation of blood flow and vessel diameter of mature and immature mice after sham or MCAO operation for 24 h. (sham: immature mice: n = 5 mice, mature mice, n = 3 mice; MCAO 24 h: immature mice: n = 5 mice, mature mice, n = 3 mice). J , representative images of five coronal slices (2 mm each) of mature and immature mice subjected to sham or MCAO 24 h followed by staining with TTC. The scale bar represents 1 mm. K , quantitation of infarct size of slices 1 to 5 or total slices in ( K ) of mature and immature mice subjected to sham or MCAO operation for 24 h (MCAO 24 h: immature mice: n = 4 mice, mature mice, n = 4 mice). Data are presented as means ± SEM (error bars). The number of repeat times ( B and D ), neurons ( F ), wells ( G ), or mice ( I and K ) analyzed are indicated in columns. Unpaired Student’s t tests ( B , D , I , and K ) and one-way ANOVA tests followed by Tukey’s multiple comparison tests ( F and G ) were performed. All experiments were repeated at least three times. LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose deprivation; TTC, 2,3,5-tetrazolium chloride.

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 tris-buffered 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, Control, Staining, Microscopy, Quantitation Assay, Comparison

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: Administration of the Tat-Syt1 T112A peptide protects against neuronal injury in cerebral ischemia. A , schematic diagram of mouse full-length Syt1 and tat peptides for Syt1 WT and Syt1 T112A . B , representative images of hippocampal neurons incubated with either Tat-V5 (control), Tat-Syt1 WT , or Tat-Syt1 T112A , followed by double immunofluorescent staining with antibodies against Syt1 or Tat-peptide. The scale bar represents 20 μm and 5 μm (magnified images). C , representative images of hippocampal neurons incubated with DMSO, Tat-V5, Tat-Syt1 WT , or Tat-Syt1 T112A followed by immunofluorescent staining with antibody against MAP2 after control or OGD treatment for 2 h. The scale bar represents 100 μm. Stained neurons were chosen randomly, and images were acquired using a confocal microscope. D and E , quantitative analysis of the average length ( D ) or the branch number ( E ) of neurons with positive MAP2 staining in ( C ). (control: DMSO: n = 82 cells/3 cultures, Tat-V5: n = 80 cells/3 cultures, Tat-Syt1 WT : n = 75 cells/3 cultures, Tat-Syt1 T112A : n = 78 cells/3 cultures; OGD 2 h: DMSO: n = 69 cells/3 cultures, Tat-V5: n = 49 cells/3 cultures, Tat-Syt1 WT : n = 54 cells/3 cultures, Tat-Syt1 T112A : n = 61 cells/3 cultures). F and G , quantitative analysis of the intracellular ATP content ( F ) and neuronal viability ( G ) of hippocampal neurons incubated with DMSO, Tat-V5, Tat-Syt1 WT , or Tat-Syt1 T112A after control or OGD treatment for 2 h. (For ATP content, control: DMSO: n = 11 wells/3 cultures, Tat-V5: n = 11 wells/3 cultures, Tat-Syt1 WT : n = 11 wells/3 cultures, Tat-Syt1 T112A : n = 11 wells/3 cultures; OGD 2 h, DMSO: n = 11 wells/3 cultures, Tat-V5: n = 11 wells/3 cultures, Tat-Syt1 WT : n = 11 wells/3 cultures, Tat-Syt1 T112A : n = 11 wells/3 cultures. For neuronal viability, control: DMSO: n = 9 wells/3 cultures, Tat-V5: n = 9 wells/3 cultures, Tat-Syt1 WT : n = 9 wells/3 cultures, Tat-Syt1 T112A : n = 9 wells/3 cultures; OGD 2 h, DMSO: n = 9 wells/3 cultures, Tat-V5: n = 9 wells/3 cultures, Tat-Syt1 WT : n = 9 wells/3 cultures, Tat-Syt1 T112A : n = 9 wells/3 cultures). H – J , representative TTC staining images ( H ), infarct volume analysis ( I ), and neurological scores ( J ) of mice with stereotactic injection of Tat-V5 or Tat-Syt1 T112A followed by sham or MCAO operation for 24 h. The scale bar represents 1 mm. (MCAO 24 h: Tat-V5: n = 5 mice: Tat-Syt1 T112A : n = 5 mice). Data are presented as means ± SEM (error bars). The number of neurons ( D and E ), wells ( F and G ), or mice ( I and J ) analyzed are indicated in columns. Unpaired Student’s t tests ( I and J ) and one-way ANOVA tests followed by Tukey’s multiple comparison tests ( D – G ) were performed. All experiments were repeated at least three times. DMSO, dimethylsulfoxide; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose deprivation; TTC, 2,3,5-tetrazolium chloride.

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 tris-buffered 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, Control, Staining, Microscopy, Injection, Comparison

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: 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 Kcnq2 WT , Kcnq2 S52A , or Kcnq2 S52D 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, Kcnq2 WT : n = 34 cells/3 cultures, Kcnq2 S52A : n = 32 cells/3 cultures, Kcnq2 S52D : n = 34 cells/3 cultures; OGD 2 h: vector: n = 32 cells/3 cultures, Kcnq2 WT : n = 21 cells/3 cultures, Kcnq2 S52A : n = 20 cells/3 cultures, Kcnq2 S52D : n = 20 cells/3 cultures). E , coimmunoprecipitation of Flag-tagged Kcnq2 and GFP-tagged Syt1 (Syt1-GFP), or GFP alone, coexpressed in HEK293T cells. (n = 3 independent repeated experiment). F and G , representative images ( F ) and quantitative analysis ( G ) of the average length of dendrites of cultured hippocampal neurons transfected with pFUGW-GFP and a vector or a plasmid encoding Syt1 or plasmids encoding Syt1 or Kcnq2, respectively, 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 = 12 cells/3 cultures, Syt1: n = 12 cells/3 cultures, Syt1+ Kcnq2: n = 25 cells/3 cultures; OGD 2 h: vector: n = 16 cells/3 cultures, Syt1: n = 14 cells/3 cultures, Syt1+ Kcnq2: n = 22 cells/3 cultures). Data are presented as means ± SEM (error bars). The number of repeat times ( B ) and neurons ( D and G ) are indicated in columns. Unpaired Student’s t tests ( B ) and one-way ANOVA tests followed by Tukey’s multiple comparison tests ( D and G ) were performed. All experiments were repeated at least three times. HEK, human embryonic kidney; Kcnq2, potassium voltage-gated channel subfamily KQT member 2; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose deprivation.

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 tris-buffered 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, Comparison

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: 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 by Western blotting. (n = 3 independent repeated experiment). B and C , representative image ( B ) and quantitation ( C ) of the GST pull assay of Kcnq2-Flag proteins bound to GST-Anxa6 or GST in the presence of 2.5 mM Ca 2+ or 4 mM EGTA, respectively, followed by Western blotting. (n = 3 independent repeated experiment). D , representative image of coimmunoprecipitation showing that Kcnq2-Flag was immunoprecipitated by GFP-tagged Anxa6 (Anxa6-GFP) in HEK293T cells. (n = 3 independent repeated experiment). E and F , representative image ( E ) and quantification ( F ) of coimmunoprecipitation of Kcnq2-Flag with Anxa6-GFP or GFP alone in the presence of 2.5 mM Ca 2+ or 4 mM EGTA, respectively, followed by Western blotting. (n = 3 independent repeated experiment). G and H , representative images ( G ) and quantitative analysis of the average length of dendrites ( H ) of hippocampal neurons transfected with pFUGW-GFP and a vector, a plasmid encoding Kcnq2, or Anxa6, or plasmids encoding Kcnq2 or Anxa6 together at DIV 10 followed by control or OGD treatment for 2 h at DIV 14. Transfected neurons were chosen randomly, and images were acquired using a confocal microscope. (control: vector: n = 41 cells/3 cultures, Kcnq2: n = 40 cells/3 cultures, Anxa6: n = 44 cells/3 cultures, Kcnq2+Anxa6: n = 43 cells/3 cultures; OGD 2 h: vector: n = 31 cells/3 cultures, Kcnq2: n = 40 cells/3 cultures, Anxa6: n = 40 cells/3 cultures, Kcnq2+Anxa6: n = 43 cells/3 cultures). I and J , representative images ( I ) and quantitative analysis of the average length of dendrites ( J ) of cultured hippocampal neurons transfected with pFUGW-GFP and a vector or a plasmid encoding Syt1, or plasmids encoding Syt1, Kcnq2, and Anxa6, respectively, together 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 = 14 cells/3 cultures, Syt1: n = 13 cells/3 cultures, Syt1+ Kcnq2+Anxa6: n = 26 cells/3 cultures, Syt1+Anxa6: n = 27 cells/3 cultures; OGD 2 h: vector: n = 15 cells/3 cultures, Syt1: n = 13 cells/3 cultures, Syt1+ Kcnq2+Anxa6: n = 26 cells/3 cultures, Syt1+Anxa6: n = 20 cells/3 cultures). Data are presented as means ± SEM (error bars). The number of repeat times ( C and F ) and neurons ( H and J ) analyzed are indicated in columns. Unpaired Student’s t tests ( C and F ) and one-way ANOVA tests followed by Tukey’s multiple comparison tests ( H and J ) were performed. All experiments were repeated at least three times. Anxa6, Annexin A6; Kcnq2, potassium voltage-gated channel subfamily KQT member 2; OGD, oxygen-glucose deprivation.

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 tris-buffered 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, Western Blot, Quantitation Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Microscopy, Cell Culture, Comparison

Journal: Cells

Article Title: Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

doi: 10.3390/cells10030567

Figure Lengend Snippet: Evidence for Ca 2+ -dependent interaction between annexin A2 (ANXA2) and S100A13 under serum-free conditions. ( A ) Pull-down assay using Strep -tagII-S100A13. Strep -tagII-S100A13 and C6 glioma cell lysates were incubated in the absence (left lane) and presence (right lane) of 100 μM Ca 2+ . Results represent the immunoblot using anti-ANXA2 IgG (upper lanes) and anti-β-actin antibody (lower lanes). The pull-down assay in A showed that ANXA2 dimer interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. In the previously published study , we have shown that p40 Syt-1 also interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. As both pull-down assays using Strep -tagII-S100A13 have been done with the same C6 glioma cell lysates, it appears that S100A13 forms the same protein complex with both experiments p40 Syt-1 and ANXA2. The experiment was done using same C6 glioma cell lysates as previously reported to show the interaction between Strep- tagII-S100A13 and p40 Syt-1 . ( B ) Serum-free-induced release of S100A13, but not ANXA2 from C6 glioma cells. Results represent the time course of protein levels of S100A13, ANXA2 and β-actin in cells (upper panels) and conditioned medium (CM) (lower panel) by immunoblot analysis. Extracellular S100A13 was recovered from CM using immunoprecipitation. ( C ) Schematic model of interaction between His 6 -ANXA2 and Strep -tagII-S100A13 bound to Streptavidin plate in an ELISA-based protein binding assay. Inset: enhancement of the interaction in the presence of His 6 -p40 synaptotagmin-1 (Syt-1). ( D , E ) No effect of amlexanox (Amx) on the His 6 -ANXA2 binding to Strep -tagII-S100A13 in terms of analyses of His 6 -ANXA2 concentration-dependency ( D ) and its reciprocal plot ( E ). ( F ) His 6 -p40 Syt-1 concentration-dependent enhancement of His 6 -ANXA2 binding to Strep -tagII-S100A13 in the presence of Ca 2+ . Data are presented as the mean ± standard error of the mean (S.E.M.) from a Tukey–Kramer multiple comparison test. * p < 0.05 and ** p < 0.01. n = 4 experiments per group.

Article Snippet: For immunoblotting, immunoprecipitation, immunocytochemistry or intracellular antibody delivery experiments, the following antibodies were used: mouse anti-prothymosin alpha (ProT α ) monoclonal antibodies (Clones: 2F11 and 4F4, Alexis Biochemicals, Lausen, Switzerland), rat anti-ProT α monoclonal antibody (Clone: 1 – 21) (developed in our laboratory, Nagasaki University, Nagasaki, Japan) [ ], N-terminus recognition goat anti-annexin A2 (ANXA2) polyclonal antibody and horseradish peroxidase (HRP)-conjugated mouse anti-β-actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-S100A13 antibody (kindly provided by Dr. T. Maciag, Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA), mouse anti-synaptotagmin-1 (Syt-1) monoclonal antibody (Wako, Osaka, Japan), mouse monoclonal anti-Stx-1 antibody (Wako, Osaka, Japan), rabbit anti-ATP8A2 polyclonal antibody (Abnova, Taipei, Taiwan), and normal mouse or goat IgG (ICN/Cappel Inc., Durham, NC, USA).

Techniques: Pull Down Assay, Incubation, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Protein Binding, Binding Assay, Concentration Assay

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A. Ablation of either Syt1 or Syt7 alone does not alter the RRP at inhibitory synapses. Hippocampal neurons were cultured from littermate WT and Syt1 KO mice (for Syt1 analysis) or from WT mice and were then infected at DIV4 with control or Syt7 KD lentiviruses (for Syt7 analysis). At DIV14–16, exocytosis of primed vesicles from the RRP in the neurons was stimulated by application of 0.5 M sucrose for 30 s (gray bars in representative traces on the left), and the RRP size was estimated as the synaptic charge transfer integrated over 30 s (summary graphs on the right). Recordings were performed in the presence of 1 μM tetrodotoxin, 20 μM CNQX, and 50 μM AP5 to isolate inhibitory currents. B. Simultaneous ablation of Syt1 and Syt7 decreases the RRP size of inhibitory synapses in a manner that is rescued by WT Syt7 (Syt1 WT ) but not Syt7 with mutations in the top C2 domain sequences containing the Syt7 Ca 2+ binding sites (Syt7 C2A * B *). Hippocampal Syt1 KO neurons were infected with control lentiviruses, Syt7 KD lentiviruses without or with expression of either WT or mutant Syt7, or lentiviruses only expressing WT Syt7 (as a control for overexpression effects). RRP was measured as described in A. C. The decreased RRP size in Syt1/7 double-deficient neurons is rescued by WT Syt1 (Syt1 WT ) but not Syt1-containing mutations in the top-loop Ca 2+ -binding sequences (Syt1 C2A * B *). Experiments were performed as described for B. All data are means ± SEM (Standard Error of the Mean); numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01; *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Cell Culture, Infection, Control, Binding Assay, Expressing, Mutagenesis, Over Expression

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A & B. Same as 1B, except that Syt7 KO neurons with a Syt1 KD were examined. Hippocampal neurons were cultured from two independent, constitutive Syt7 KO mouse lines (D, Syt7 KO A from ; E, Syt7 KO N from ), and infected with control lentivirus or Syt1 KD lentivirus without or with superinfection with a second lentivirus expressing WT or mutant Syt7. All data are means ± SEM; numbers in bars indicate number of neurons or independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01; *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Cell Culture, Infection, Control, Expressing, Mutagenesis

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A & B. Ablation of both Syt1 and Syt7 in hippocampal neurons decreases the RRP size at excitatory synapses in a manner that is rescued by WT Syt7 (Syt7 WT ) or Syt1 (Syt1 WT ) but not by mutant Syt7 (Syt7 C2A * B *) or Syt1 (Syt1 C2A * B *) with altered top-loop sequences containing the Ca 2+ -binding sequences (A, Syt7 rescue; B, Syt1 rescue). Experiments were performed as described for , except that picrotoxin (50 μM) was used instead of CNQX. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01, *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Mutagenesis, Binding Assay

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A. Representative electron micrographs from cultured hippocampal Syt1 KO neurons that were infected with control lentivirus or Syt7 KD lentiviruses without or with expression of WT or mutant Syt7 rescue cDNAs. B. Quantification of the total number of docked vesicles (defined as vesicles touching the membrane), vesicles at the active zone (AZ, defined as vesicles within 100 nm of plasma membrane), total number of vesicles in each bouton, and postsynaptic density (PSD) length and bouton area in EM micrographs. Quantification from two independent cultures was performed manually by an observer blind to the conditions. No significant differences were found for the parameters analyzed. The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Cell Culture, Infection, Control, Expressing, Mutagenesis, Membrane, Clinical Proteomics

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A. In Syt1/7 double-deficient neurons, Syt1 mutations in either the C2A-domain (Syt1 C2A *) or C2B domain (Syt1 C2B *) top-loop sequences that include their Ca 2+ binding sites do not block rescue of the RRP in Syt1/7 double deficient neurons, whereas Syt7 mutations in the C2A-domain (Syt1 C2A *) but not the C2B domain (Syt1 C2B *) Ca 2+ -binding sequence selectively impair rescue of the RRP. Hippocampal neurons were cultured from Syt1 KO mice and infected with control lentivirus, or Syt7 KD lentiviruses co-expressing the indicated Syt1 and Syt7 mutants. Exocytosis of primed vesicles from the RRP was induced by a brief pulse of 0.5 M sucrose, and monitored as IPSCs in the presence of TTX (1 μM), CNQX (20 μM), and AP5 (50 μM). Representative traces are shown on the left, and summary graphs on the right. Note that simultaneous mutation of both Syt1 C2-domains blocks its priming function . B. Confirmation that mutations of Syt1 in the top-loop sequences containing the Ca 2+ binding sites of the C2B domain but not the analogous mutations in the C2A domain block rescue of synchronous release in Syt1 KO neurons. Experiments were performed as described for A, except that IPSCs were elicited by 10 Hz, 1 s stimulus trains (left, representative traces; right, summary graphs of the total synaptic charge transfer). Stimulations are indicated by tick marks. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Binding Assay, Blocking Assay, Sequencing, Cell Culture, Infection, Control, Expressing, Mutagenesis

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A & B. Analysis of the SNARE binding of WT Syt1 and Syt7 (Syt1 WT and Syt7 WT , respectively) and of mutant Syt1 and Syt7-containing top-loop substitutions altering their Ca 2+ -binding sequences (Syt1 C2A * B * and Syt7 C2A * B *, respectively; A, Syt1 WT and Syt1 C2A * B *; B, Syt7 WT and Syt7 C2A * B *). WT or mutant Syt1 and Syt7 were lentivirally expressed in neurons, and neurons were analyzed at DIV14–16 by immunoprecipitation of the SNARE protein syntaxin-1 followed by immunoblotting for the SNARE protein synaptobrevin-2 (Syb2; to assess the degree of SNARE complex IP) or of Syt1 or Syt7 (to assess the degree of synaptotagmin binding). Data are means ± SEM ( n = 3 independent culture experiments). Statistical significance was assessed by one-way ANOVA (*** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Western Blot

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A. Syt1 alone has no effect on SNARE complex assembly. HEK293T cells were cotransfected with plasmids expressing syntaxin-1 (Synt-1), SNAP-25, and synaptobrevin-2 (Syb-2) in a 1:1:1 ratio, together with increasing amounts of Syt1 expression plasmid (0 to 4-fold), and decreasing amounts of emerald expression plasmid (GFP; 4 to 0-fold), to balance the total amount of transfected DNA. Cell lysates were immunoblotted for Syt1, emerald, SNARE complexes (high molecular mass bands), and total SNARE proteins, followed by quantitation ( n = 4 independent experiments). B. Complexin increases SNARE complex assembly. Same as (A), except that HEK293T cells were co-transfected with an increasing amount of complexin-1 (Cpx1) expression plasmid ( n = 4 independent experiments). All data are means ± SEM; statistical significance was assessed by Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Expressing, Plasmid Preparation, Transfection, Quantitation Assay

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: Syt1 increases SNARE complex assembly in presence of complexin-1. HEK293T cells were cotransfected with plasmid expressing Synt-1, SNAP-25, Syb-2, and complexin-1 in a 1:1:1:3 ratio, together with an increasing amount of Syt1 expression plasmid (0 to 4-fold), and decreasing amount of emerald expression plasmid (GFP; 4 to 0-fold). Cell lysates were immunoblotted for Syt-1, Cpx1, emerald, SNARE complexes (high molecular mass bands), and total SNARE proteins followed by quantitation ( n = 6 independent experiments). All data are means ± SEM; statistical significance was assessed by Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Plasmid Preparation, Expressing, Quantitation Assay

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A & B. Simultaneous ablation of Syt1 and Syt7 impairs the RRP size but not the relative rate of RRP recovery after RRP depletion at inhibitory synapses. In A, Syt1 KO neurons without or with induction of the Syt7 KD alone or together with the Syt7 rescue were analyzed; in B, Syt7 KO neurons (from KO strain A) without or with Syt1 KD were tested. The RRP was measured during an initial 10 s application of 0.5 M sucrose which also depleted the RRP, and RRP recovery was monitored after 40 s by a second 10 s application of sucrose (gray bars = sucrose applications). Double deficiency of Syt1 and Syt7 resulted in a reduced RRP size with no effect on the relative rate of RRP recovery (left, representative traces; middle, summary graphs of absolute RRP sizes; right, summary graphs of the ratio of 2nd versus 1st RRP to estimate RRP recovery). Recorded currents were not corrected for the significant changes in access resistance similarly observed for all groups (see ). All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (*** p < 0.001). The data used to make this figure can be found in .

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Relative Rate

Journal: PLoS Biology

Article Title: Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

doi: 10.1371/journal.pbio.1002267

Figure Lengend Snippet: A. Schematic diagram of the different stages of Ca 2+ -triggered synaptic vesicle exocytosis and of the functions of Syt1 and Syt7 in the maintenance of the RRP size, clamping spontaneous mini release (which is in itself Ca 2+ -dependent via the actions of an as yet unidentified Ca 2+ sensor ), and Ca 2+ triggering of delayed and fast release. B. The three different functions of Syt1 (top) and Syt7 (bottom) are depicted by arrows; the C2 domain requirement for each function is described on top of the arrows. In addition, schematic diagrams of the Syt1 and Syt7 domain structures are shown, with the top-loop Ca 2+ -binding sequences of a given C2 domain that is required for a particular function indicated in red.

Article Snippet: Coprecipitated proteins were separated by SDS-PAGE followed by detection with monoclonal antibodies against an HA epitope included in Syt1 (HA.11; 16B12, Covance) and synaptobrevin-2 (cl.

Techniques: Binding Assay