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