mouse anti syt 1  (Cell Signaling Technology Inc)

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: bioRxiv

Article Title: Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

doi: 10.1101/704205

Figure Lengend Snippet: A. Immunolabeling was performed in WT cells for endogenous Syt-7 (red) and Syt-1 (green). Images from a representative cell showing Syt-7 fluorescence, Syt-1 fluorescence, and fluorescence of the merged channels. B. Bar graphs indicate the percentage of Syt-1 puncta that harbor Syt-7 (5.18 ± 1.03 %; n = 8 cells) and Syt-7 puncta that harbor Syt-1 (3.36 ± 0.43 %; n = 8 cells). C. Immunolabeling was performed in WT cells for Syt-7 (green) and PAI-1, a dense core vesicle marker (red). D. Bar graphs showing the percentage of PAI-1 puncta that are co-localized with Syt-7 (24.06 ± 1.41 %; n = 8 cells) and the percentage of Syt-7 puncta that are co-localized with PAI-1 (14.65 ± 1.075 %; n = 8 cells).

Article Snippet: Cat # 105 173, 1:1200), and monoclonal mouse anti-Syt-1 (Synaptic Systems, Göttingen, Germany.

Techniques: Immunolabeling, Fluorescence, Marker

Journal: bioRxiv

Article Title: Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

doi: 10.1101/704205

Figure Lengend Snippet: A . Expression of Syt-1, NPY, tPA, and TH transcripts in WT and Syt-7 KO chromaffin cells. WT and KO adrenal medullas were homogenized, mRNA was extracted and RT-qPCR was performed on the samples for Syt-1 (n = 5), NPY (n = 3), tPA (n = 3), TH (n = 3), and Syt-7 (n = 2). Syt-7 transcript was not detected using the primers shown in Table 1. Expression of Syt-1 mRNA was not significantly different in WT and KO cells (Student’s t-test, p > 0.05). B. Western Blot analysis was performed on WT and Syt-7 KO adrenal glands. Boxed region (white) indicates the region where the approximately 45 kDa Syt-7 alpha variant should be observed ( , ). It is present in the WT lanes but not in the Syt-7 KO lanes.

Article Snippet: Cat # 105 173, 1:1200), and monoclonal mouse anti-Syt-1 (Synaptic Systems, Göttingen, Germany.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Variant Assay

Journal: bioRxiv

Article Title: Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

doi: 10.1101/704205

Figure Lengend Snippet: A-B. Fusion of dense core vesicles containing either Syt-1 or Syt-7 with planar supported bilayers containing syntaxin-1a, dSNAP-25, Munc18, and complexin-1 were examined. As previously described, dense core vesicles labeled with NPY-mRuby stably dock to the supported bilayer, where fusion is triggered by injection of calcium. C. The calcium sensitivity of fusion is dependent on the Sytisoform expressed on purified dense core vesicles (K 1/2 = 63 ± 3 μM for Syt-1 vesicles and 10 ± 1 μM for Syt-7 vesicles). Example of a characteristic NPY-mRuby signal D-E. is observed for fusion. There is an initial decrease in NPY-mRuby fluorescence caused by diffusion of the protein away from the site of fusion under the cleft of the supported bilayer. This is followed by an increase in fluorescence caused by a collapse of the vesicle into the supported bilayer. This action pulls the remaining lumenal content towards the brightest portion of the evanescent field. Eventually, the fluorescence diminishes as NPY-mRuby diffuses away from the site of fusion. Averaging more than 20 individual traces (D, E) for Syt-1 fusion events, revealed a short delay (D) between the initial decrease in fluorescence and the subsequent increase in fluorescence during vesicle collapse. The delay time between these phases of the fluorescent signal was prolonged for Syt-7 vesicle fusion. The cartoons illustrate the mathematical model (solid line in C and D) used to fit the data.

Article Snippet: Cat # 105 173, 1:1200), and monoclonal mouse anti-Syt-1 (Synaptic Systems, Göttingen, Germany.

Techniques: Labeling, Stable Transfection, Injection, Purification, Fluorescence, Diffusion-based Assay

Journal: bioRxiv

Article Title: Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

doi: 10.1101/704205

Figure Lengend Snippet: A-B. Chromaffin cell vesicles harbor Syt-1, Syt-7, or both (not shown). When stimulated with ACh, intracellular calcium concentrations increase rapidly , leading to fusion of Syt-1 and Syt-7 vesicle popula-tions. C. Prolonged stimulation with ACh causes nicotinic desensitization. Associated with desensitization is a corresponding decline in intracellular Ca 2+ which is buffered, sequestered, or otherwise removed from the cytosol ( and ). Because Syt-1 has a lower affinity for Ca 2+ than Syt-7, vesicles bearing this isoform are less likely to fuse as intracellular Ca 2+ levels decline. On the other hand Syt-7-bearing vesicles continue to fuse even with diminished free cytosolic Ca 2+ .

Article Snippet: Cat # 105 173, 1:1200), and monoclonal mouse anti-Syt-1 (Synaptic Systems, Göttingen, Germany.

Techniques: