Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Expression of SNARE STX8 in platelets, its localization and interactions. A , resting human and mouse platelets were lysed in SDS sample buffer and proteins were separated on SDS-PAGE. The resulting blots were probed with antibodies as shown. B, human double washed platelets were solubilized using digitonin as described. The resulting digitonin-soluble ( cytosol ) and digitonin-insoluble ( membrane ) fractions were separated on SDS-PAGE and blotted as before and localization of STX8 was compared with localization of Fc receptor γ signaling chain ( FcR γ, membrane marker ), GAPDH (cytosol marker), and STX11 (membrane-associated SNARE). C, human resting ( R ) and activated ( A , 1 unit/ml of thrombin) double washed platelets were lysed as described and proteins were immunoprecipitated ( IP ) with relevant antibodies. The resultant immune complexes were denatured, separated on SDS-PAGE, and blotted as before. Rabbit nonspecific Ig controls (same species as IP antibodies) were also included. D, the STX8-STX11 complex identified was found to be constitutive, with no significant change upon activation as quantified using ImageJ ( n = 4). Data shown ( A–C ) are representative of at least 3 experiments, error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Expressing, SDS Page, Membrane, Marker, Immunoprecipitation, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Hematology parameters of Stx8 −/− mice Hematology parameters were measured in whole anticoagulated blood (adjusting for the volume of anticoagulant). There was no difference in platelet count, mean platelet volume (MPV), red blood cell count (RBC), or white blood cell counts (WBC) between the genotypes (ANOVA, p > 0.05).

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Initial characterization of Stx8 −/− platelets. A, resting platelets were prepared for TEM imaging, and images were acquired using Tecnai-12 electron microscope at ×4300 magnification. Arrows denote α granules, arrowheads denote dense granules ( scale bar : 1 μm). Images are representative of 3 independent observations. B, the levels of glycoprotein expression were measured using FACS and compared with expression levels of WT platelets. There was no significant difference between the genotypes ( n = 5, p > 0.05, two-way ANOVA). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Imaging, Microscopy, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Quantification of granule secretion in Stx8 −/− platelets. All concentration-response data were analyzed by appropriate curve fitting in GraphPad Prism. A, mouse platelets were stimulated in the aggregometer as described. Dense granule secretion was measured as an increase in luminescence relative to ATP standard for each stimulation. Data were normalized to maximum ATP secretion of WT platelets on the day of experiment. There was a significant increase in [thrombin] EC 50 in Stx8 −/− (95% CI of EC 50 : 0.085 to 0.11 units/ml) compared with WT platelets (95% CI of EC 50 : 0.068 to 0.086 units/ml) ( n = 5, variable slope sigmoidal dose-response curve, p = 0.0248). Stx8 −/− and WT secretion in response to 0.06 units/ml was, respectively, 1.742 ± 0.618 and 10.379 ± 3.767% of the maximum WT secretion on the day ( n = 4, p > 0.05, ns), and in response to 0.075 units/ml of thrombin: 6.927 ± 5.970 and 38.933 ± 20.969% of the maximum WT secretion, p = 0.0339). B, the total 5-HT content was measured using 3 H-labeled 5-HT as described. There was no significant difference in 5-HT content between genotypes ( n = 3). C, α-granule secretion was measured by FACS. Median fluorescent intensity (MFI) of FITC-CD62P was quantified in response to increasing concentrations of thrombin. MFI was normalized to the maximum MFI of WT platelets on the day of experiment ( n = 4). D, lysosome secretion was measured by β-hexosaminidase enzyme activity in supernatants following stimulation with increasing concentrations of thrombin. Absorbance at 405 nm was measured, and values were expressed as percentage of TOTAL control for each subject ( n = 6). There was no difference in [thrombin] EC 50 between genotypes in α-granule or lysosome secretion ( p > 0.05, variable slope sigmoidal dose-response curve). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Concentration Assay, Labeling, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Analysis of aggregation and ADP rescue experiments in Stx8 −/− platelets. A, aggregation was measured using lumi-aggregometer and amplitude was expressed as percentage decrease in optical density of the sample at t = 180 s ( n = 4). The concentration-response curve for Stx8 −/− was shifted to the right, but [thrombin] EC 50 was not significantly different (95% CI of WT EC 50 : 0.042 to 0.090, 95% CI of Stx8 −/− EC 50 : 0.069 to 0.101, p > 0.05). However, the maximum aggregation amplitude was significantly reduced in Stx8 −/− at 0.075 units/ml of thrombin (26.1 ± 13.6% decrease in optical density in Stx8 −/− versus 60.8 ± 18.2% in WT, p = 0.021, two-way ANOVA with Bonferroni post-test), with aggregation at 0.05 and 0.06 units/ml also consistently reduced, but not significantly. B, integrin α IIb β 3 activation was measured by FACS, no difference in PE-JON/A binding was observed between genotypes suggesting an alternative mechanism for the observed aggregation defect. C, representative aggregation traces showing the effect of co-stimulation with 10 μ m exogenous ADP response to EC 50 concentration of thrombin ( i ) and CRP ( ii ). D, aggregation in response to co-stimulation with ∼EC 50 (0.05–0.075 units/ml) thrombin and 10 μ m ADP was compared with EC 50 thrombin alone ( n ≥ 5). Stx8 −/− aggregation was significantly reduced in response to thrombin alone (37.8 ± 8.9 versus 63.4 ± 6.1% decrease in optical density, respectively, p < 0.05) and could be fully rescued with ADP ( p > 0.05). The maximum aggregation to 1 unit/ml of thrombin was the same between genotypes (data not shown). Similarly, the effect of co-stimulation with ADP and EC 50 CRP (0.3–0.5 μg/ml) was measured ( n ≥ 4). There was a significant reduction in aggregation in response to ∼EC 50 CRP in Stx8 −/− platelets (30.0 ± 7.3 versus 46.3 ± 8.6% in WT, p < 0.05) that was fully rescued with ADP ( p > 0.05). Maximum response (to 5 μg/ml of CRP) was the same (data not shown). E, secretion of ATP was also measured. Again, secretion in response to EC 50 concentrations of thrombin or CRP was significantly reduced ( p < 0.01), whereas co-stimulation with ADP could only partially rescue secretion when thrombin was used as primary agonist ( p < 0.05). In the case of co-stimulation with CRP, ADP enhanced ATP secretion to a much greater extent in both WT and Stx8 −/− platelets (ADP enhancement of 185.75 ± 42.55% of CRP alone, compared with 111.30 ± 20.66% of thrombin alone). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Concentration Assay, Activation Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Analysis of secretion in Vti1b −/− platelets. A, expression levels of other known SNAREs and SNARE-associated proteins were measured in Stx8 −/− platelets to ensure the defect results from Stx8 deletion. Levels of STX11, VAMP8, SNAP23, and MUNC13d and 18b were comparable between the genotypes (image representative of at least 3 independent experiments). B, a significant decrease in expression of Vti1b in Stx8 −/− platelets was observed (31.13 ± 5.85% of WT expression, p < 0.01). C, similarly, Stx8 was also down-regulated in Vti1b −/− platelets. D, to confirm that only complete ablation of Stx8 leads to defective dense granule secretion, we analyzed ATP secretion from Stx8 +/− platelets and found no defect, unlike in Stx8 −/− platelets ( n > 3, difference not significant but consistent for WT versus Stx8 −/− , two-way ANOVA). E–G, to further confirm that deletion of Stx8 and not the associated down-regulation of Vti1b was responsible for defect in dense granule secretion observed, we measured secretion from dense ( E ) and α-granules ( F ) and lysosomes ( G ) in Vti1b −/− platelets. No difference was observed in any of the secretion events in Vti1b −/− platelets ( n = 6). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: Analysis of in vitro thrombus formation under shear in Stx8 −/− whole blood. Anti-coagulated whole blood was flowed over immobilized collagen (50 μg/ml) or fibrinogen (100 μg/ml) through a parallel plate perfusion chamber at a shear rate of 1000 s −1 for 4 min. A and B, the chamber was constantly imaged over the time of experiment and the percentage coverage at each time point was acquired using QCapture software. The change in coverage represents mean ± S.E. ( n ≥ 4). C, at the end of each experiment, chambers were washed for 3 min with buffer to remove non-adherent platelets and erythrocytes, and 30 random images of the whole coverslip were taken. The surface area covered by thrombi was analyzed using ImageJ ( n ≥ 4). There was no difference in either the rate of adhesion or the size of thrombi in Stx8 −/− , either on collagen (GPVI-mediated adhesion) or on fibrinogen (α IIb β 3 -mediated adhesion). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: In Vitro, Shear, Software

Journal: The Journal of Biological Chemistry

Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *

doi: 10.1074/jbc.M114.602615

Figure Lengend Snippet: In vivo thrombus formation and embolization of analysis in Stx8 −/− and WT animals. Carotid artery damage was achieved by treatment with FeCl 3 as described. Fluorescently labeled platelets adhering at the site of injury could then be imaged continuously. A, the rate of platelet adhesion was slower in Stx8 −/− , but the difference was not significant (adhesion rate shown as mean ± S.E.). B, the final thrombi size (expressed as IFD of the field of view) at the end of a 20-min experiment was also reduced in Stx8 −/− animals but the difference was not significant. The videos were then reviewed again, focusing on the number of embolization events as shown in C . An embolus, seen here as a small fragment of the shell of the growing thrombus ( arrow at 0 s) becomes dislodged and initially “rolls” with the direction of blood flow (at 5 s) to eventually become detached from the thrombus and carried away by the blood flow (6 s). D, when counted throughout the duration of the experiment, the number of embolization events was significantly increased in the Stx8 −/− (137.3 ± 20.12) compared with WT animals (75.46 ± 17.71) ( n = 8, p = 0.037, t test). E, mean time to cessation of bleeding in tail bleeding assay was the same in Stx8 −/− and WT animals ( t < 180 s, n = 6). Error bars represent S.E.

Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).

Techniques: In Vivo, Labeling

Journal: Aging Cell

Article Title: Extra virgin olive oil improves synaptic activity, short‐term plasticity, memory, and neuropathology in a tauopathy model

doi: 10.1111/acel.13076

Figure Lengend Snippet: Effect of chronic administration of EVOO‐rich diet on hippocampal pre‐ and postsynaptic protein expression in hTau mice. (a) Representative Western blot analyses for SNAP25, syntaxin 1A, synaptobrevin, complexin 1/2, and synaptophysin (SYP) in hippocampus homogenates from CTR and EVOO mice. (b) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (** p < .01; n = 6 per group) . (c) Representative Western blot analyses for the AMPAR subunit glutamate receptor 1 (GluR1), AMPAR subunit glutamate receptor 2 (GluR2), and their phosphorylated forms, pGluR1 (S831), pGluR1 (S845), and pGluR2 (S880), in hippocampus homogenates from CTR and EVOO mice. (d) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A ( n = 6 per group) . Values are mean ± SEM

Article Snippet: Syntaxin 1A , AF7237 , RD SYSTEMS , Goat.

Techniques: Expressing, Western Blot

Journal: Aging Cell

Article Title: Extra virgin olive oil improves synaptic activity, short‐term plasticity, memory, and neuropathology in a tauopathy model

doi: 10.1111/acel.13076

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Syntaxin 1A , AF7237 , RD SYSTEMS , Goat.

Techniques:

Journal: Journal of ginseng research

Article Title: Fructose-arginine, a non-saponin molecule of Korean Red Ginseng, attenuates AIM2 inflammasome activation.

doi: 10.1016/j.jgr.2020.06.002

Figure Lengend Snippet: Fig. 1. Effects of RGE, NS, and SF on AIM2 inflammasome activation. A, Schematic diagram of the experimental process used for inflammasome activation in macrophages. B, Mouse BMDMs were primed with LPS and then subjected to dsDNA transfection to activate AIM2 inflammasome in the presence of RGE, NS, or SF as indicated. IL-1b secretion was analyzed by immunoblot assay. C, LPS-primed BMDMs were treated with NG along with increasing dosages of NS or SF, as indicated, to trigger NLRP3 inflammasome activation. The release of IL-1b was observed by immunoblot assay. All data shown are representative of at least two independent experiments. RGE, Korean Red Ginseng extracts; NS, non-saponin fraction of RGE; SF, saponin fraction of RGE; Sup, cellular supernatant; Lys, cellular lysate.

Article Snippet: The membranes were blocked by 3 % skim milk and probed with anti-mouse IL-1b antibody (R&D Systems, MN, USA), anti-caspase-1 antibody (AdipoGen Co., San Diego, CA, USA), anti-Asc antibody (Santa Cruz Biotechnology), anti-GSDMD antibody (Abcam plc., Cambridge, MA, USA), or anti-actin antibody (Santa Cruz Biotechnology) overnight at 4 C. The membranes were additionally probed with secondary antibody conjugated with HRP against the first anti-sera (Santa Cruz Biotechnology) for 3 h at room temperature and then visualized by applying an enhanced chemiluminescence solution (WESTSAVE STAR , AbFrontier, Seoul, Republic of Korea) and using a chemiluminometer (EZ-Capture II, ATTO Technology, Tokyo, Japan).

Techniques: Activation Assay, Transfection, Western Blot

Journal: Journal of ginseng research

Article Title: Fructose-arginine, a non-saponin molecule of Korean Red Ginseng, attenuates AIM2 inflammasome activation.

doi: 10.1016/j.jgr.2020.06.002

Figure Lengend Snippet: Fig. 2. Effects of SEF on AIM2 inflammasome activation in macrophages and mice. A, Mouse BMDMs were primed with LPS for 3 h and then treated with dsDNA for AIM2 inflammasome activation in the presence of increasing concentrations of SEF as indicated. The secretion of caspase-1 (Casp1) was analyzed by immunoblot assay and the release of IL-1b was determined by ELISA. B and C, PMA-treated THP-1 cells (human macrophage-like cell line) were primed with LPS for 3 h and then subjected to dsDNA transfection in the presence of SEF or FA (fructose-arginine) as indicated. D, LPS-primed BMDMs were inoculated with Listeria monocytogenes (LM) to activate AIM2 inflammasomes. IL-1b releases were analyzed by ELISA. E, Mice (n ¼ 6 per group) were fed daily with SEF, as indicated, for 7 d, and then intraperitoneally injected with 200 mL of PBS containing LM. After 6 h, peritoneal lavage and blood serum samples were collected, and IL-1b secretions were measured by ELISA. Bar graphs indicate mean SD values. All data shown are representative of at least two independent experiments.

Article Snippet: The membranes were blocked by 3 % skim milk and probed with anti-mouse IL-1b antibody (R&D Systems, MN, USA), anti-caspase-1 antibody (AdipoGen Co., San Diego, CA, USA), anti-Asc antibody (Santa Cruz Biotechnology), anti-GSDMD antibody (Abcam plc., Cambridge, MA, USA), or anti-actin antibody (Santa Cruz Biotechnology) overnight at 4 C. The membranes were additionally probed with secondary antibody conjugated with HRP against the first anti-sera (Santa Cruz Biotechnology) for 3 h at room temperature and then visualized by applying an enhanced chemiluminescence solution (WESTSAVE STAR , AbFrontier, Seoul, Republic of Korea) and using a chemiluminometer (EZ-Capture II, ATTO Technology, Tokyo, Japan).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Injection

Journal: Journal of ginseng research

Article Title: Fructose-arginine, a non-saponin molecule of Korean Red Ginseng, attenuates AIM2 inflammasome activation.

doi: 10.1016/j.jgr.2020.06.002

Figure Lengend Snippet: Fig. 3. Effects of fructose-arginine on AIM2 inflammasome activation. A, LPS-primed BMDMs were transfected with dsDNA in the presence of SEF (100 mg/mL), arginine (1 mg/mL), fructose (1 mg/ml), glucose (1 mg/mL), or maltose (1 mg/mL). Secretions of caspase-1 (Casp1) and IL-1b were measured by immunoblot assay and ELISA, respectively. B, BMDMs were primed with LPS, and AIM2 inflammasomes were activated by dsDNA transfection in the presence of the amino-sugars fructose-arginine (FA) or arginine-fructose-glucose (AFG). C, LPS-primed BMDMs were transfected with dsDNA to activate AIM2 inflammasomes. Immunoblot assays were conducted to detect secretions of IL-1b and caspase-1 (Casp1), the formation of Asc oligomerization, and the cleavage of gasdermin D (Gsdmd), as indicated. The secretions of IL-1b and IL-18 were measured by ELISA. Bar graphs indicate mean SD values. All data shown are representative of at least two independent experiments.

Article Snippet: The membranes were blocked by 3 % skim milk and probed with anti-mouse IL-1b antibody (R&D Systems, MN, USA), anti-caspase-1 antibody (AdipoGen Co., San Diego, CA, USA), anti-Asc antibody (Santa Cruz Biotechnology), anti-GSDMD antibody (Abcam plc., Cambridge, MA, USA), or anti-actin antibody (Santa Cruz Biotechnology) overnight at 4 C. The membranes were additionally probed with secondary antibody conjugated with HRP against the first anti-sera (Santa Cruz Biotechnology) for 3 h at room temperature and then visualized by applying an enhanced chemiluminescence solution (WESTSAVE STAR , AbFrontier, Seoul, Republic of Korea) and using a chemiluminometer (EZ-Capture II, ATTO Technology, Tokyo, Japan).

Techniques: Activation Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay