Journal: Molecular & cellular proteomics : MCP

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

doi: 10.1016/j.mcpro.2022.100222

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

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

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

Journal: Molecular & cellular proteomics : MCP

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

doi: 10.1016/j.mcpro.2022.100222

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

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

Techniques: Incubation

Journal: Molecular & cellular proteomics : MCP

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

doi: 10.1016/j.mcpro.2022.100222

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

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

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

Journal: Molecular & cellular proteomics : MCP

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

doi: 10.1016/j.mcpro.2022.100222

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

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

Techniques: Pull Down Assay