Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 3 Levels of neuronal markers were not altered in 18-month-old AppNL-F/wt knock-in; ob/ob mice. Levels of NF-H (A), SYT1 (B), PSD95 (C), and phospho-tau (D) were assessed using ELISAs and compared among each genotype after adjusting for sex (n = 10-44 mice/group). Standard curve of ELISAs of NF-H (E), SYT1 (F), PSD95 (G), and phospho-tau (H) with sample range as shown in red line in a representative assay. A-D, Data are presented as adjusted means ± standard errors of the means and were compared among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 5 Levels of neuronal and glial markers in young (6-month-old) AppNL-F/wt knock-in; ob/ob mice. A, Body weight of young mice were compared among each genotype after adjusting for sex (n = 8-17 mice/group). B-F, Levels of NF-H (B), SYT1 (C), PSD95 (D), CD11b (E), and GFAP (F) in the brains of young mice were compared among each genotype after adjusting for sex (n = 9-17 mice/group). Data are presented as adjusted means ± standard errors of the means. *P < .05, **P < .01, and ***P < .001 for the comparisons among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

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: The Journal of Cell Biology

Article Title: The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export

doi: 10.1083/jcb.200203084

Figure Lengend Snippet: Dominant-negative mutants of S100A13 and Syt1 do not affect the heat shock–induced redistribution of cytosolic FGF1:GFP. (A) NIH 3T3 cells were transiently cotransfected with FGF1:GFP and either S100A13:Myc (a–f) or S100A13Δ88–98:Myc (g–l), and 48 h later the cells were fixed after a 2-h incubation at either 37 (a–c and g–i) or 42°C (d–f and j–l). The cells were stained with an anti-Myc antibody followed by an Alexa-647–conjugated secondary antibody. Confocal images of median horizontal cell sections were taken using the 100X objective and the intracellular distribution of FGF1:GFP (a, d, g, and j), S100A13:Myc (b and e), S100A13Δ88–98Myc (h and k), and their respective overlays (c, f, i, and l) are shown. Bar, 10 μM. (B) NIH 3T3 cells were transiently cotransfected with FGF1:GFP and either Syt1:Myc (a–f) or Syt1Δ120–214:Myc (g–l) and 48 h later the cells were fixed after 2 h of incubation at either 37 (a–c and g–i) or 42°C (d–f and j–l). The cells were stained with either an anti-Syt1 (b and e) or an anti-Myc (h and k) antibody followed by an Alexa 647–conjugated secondary antibody. Confocal images of median horizontal cell sections were taken using the 100× objective and the intracellular distribution of FGF1:GFP (a, d, g, and j), Syt1:Myc (b and e), Syt1Δ120–214:Myc (h and k), and their respective overlays (c, f, i, and l) are shown. Bar, 10 μM.

Article Snippet: A monoclonal antibody to the extravesicular fragment of Syt1 (Synaptic Systems) followed with an Alexa 647–conjugated anti–mouse IgG antibody was used to detect wild-type Syt1 and rabbit antibodies against Erk1 and Erk2 were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Dominant Negative Mutation, Incubation, Staining

Journal: EMBO Reports

Article Title: Synaptotagmin-11 facilitates assembly of a presynaptic signaling complex in post-Golgi cargo vesicles

doi: 10.1038/s44319-024-00147-0

Figure Lengend Snippet: ( A ) Representative confocal images of HEK293T cells expressing GB2-VC and Syt11-VN tagged with the C-terminal (VC) and N-terminal (VN) fragments of the fluorescent Venus protein (top row). Reconstitution of Venus fluorescence is observed only in cells expressing KCTD16. In control experiments, replacing Syt11-VN with Syt11ΔC2-VN lacking the C2A and C2B domains (middle row) or Syt1-VN (bottom row) does not reconstitute Venus fluorescence. Transfected cells were identified using mCherry. Scale bar: 10 μm. ( B ) Representative Western blots (left) and corresponding quantifications from n = 5 independent experiments (right) of APs with anti-HA antibodies from cell lysates of transfected HEK293T cells expressing the indicated constructs. AP and input lanes were probed with anti-Syt11 (top), anti-KCTD16 (middle), and anti-HA (bottom) antibodies. The presence of VN- or VC-tags on Syt11 and GB2, respectively, does not significantly alter the amounts of KCTD16 ( p = 0.436) and Syt11 ( p = 0.858) co-purified with GB2. Values are presented as mean ± SEM, ns = not significant, unpaired t-test.

Article Snippet: The Syt1 plasmid was from OriGene (Cat# MR206688), pcDNA3.1-mCherry from Addgene (Cat# 128744), NPY-mCherry from Addgene (Cat# 67156) and pSI-AAR6-Rab5a-mcherry was a gift from M. Spiess (Kalin et al, ).

Techniques: Expressing, Fluorescence, Control, Transfection, Western Blot, Construct, Purification

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: 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