Figure 1.
SNARE machinery proteins levels remain unchanged in CSPα−/− platelets. (A) Washed platelets (50 x 103/µL platelets per lane) were prepared from CSPα+/+, CSPα+/−, and CSPα−/− mice (n = 4), and the indicated proteins were probed by western blotting. Data are representative of 4 independent experiments. Because of molecular weight overlaps between probed proteins and loading controls, different controls were on different blots. Blots for CSPα, α-synuclein, and RabGDI were on the same membrane (RabGDI as control). VAMP-8, SNAP-23, and RabGDI were on the same membrane (RabGDI as control). Syntaxin-17 and RabGDI were on the same membrane (RabGDI as control). VAMP-3, VMAT2, and syntaxin-11 were on the same membrane (syntaxin-11 as control). Fibrinogen and syntaxin-11 were on the same membrane (syntaxin-11 as control). Platelet factor 4 and actin were on the same membrane (actin as control). GPVI and actin were on the same membrane (actin as control). (B) Quantification of protein levels was performed using ImageLab, and data were plotted as the ratio of CSPα+/− to CSPα+/+ (gray bars) and CSPα−/− to CSPα+/+ (red bars). Statistical analyses were done using individual values and performed using the unpaired nonparametric Mann-Whitney U test. (C) Washed platelets (50 x 103/µL) from CSPα+/+, CSPα+/−, and CSPα−/− were resting and incubated with fluorescein isothiocyanate–conjugated anti-GPVI antibodies for 20 minutes at 37°C. Fluorescent intensities were measured by flow cytometry. Shown are representative data and geometric mean fluorescent intensity (GMFI) (mean ± standard error) of 5 independent experiments. Statistical analyses were performed using the Kruskal-Wallis multiple comparison test and corrected using the Dunn multiple comparison test. Significance: ns = P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. All error bars represent the standard error of the mean. AU, arbitrary units; HET, heterozygous; KO, knockout; ns, not significant; RabGDI, rab guanosine diphosphate dissociation inhibitor; VMAT2, vesicle monoamine transporter 2.

SNARE machinery proteins levels remain unchanged in CSPα−/− platelets. (A) Washed platelets (50 x 103/µL platelets per lane) were prepared from CSPα+/+, CSPα+/−, and CSPα−/− mice (n = 4), and the indicated proteins were probed by western blotting. Data are representative of 4 independent experiments. Because of molecular weight overlaps between probed proteins and loading controls, different controls were on different blots. Blots for CSPα, α-synuclein, and RabGDI were on the same membrane (RabGDI as control). VAMP-8, SNAP-23, and RabGDI were on the same membrane (RabGDI as control). Syntaxin-17 and RabGDI were on the same membrane (RabGDI as control). VAMP-3, VMAT2, and syntaxin-11 were on the same membrane (syntaxin-11 as control). Fibrinogen and syntaxin-11 were on the same membrane (syntaxin-11 as control). Platelet factor 4 and actin were on the same membrane (actin as control). GPVI and actin were on the same membrane (actin as control). (B) Quantification of protein levels was performed using ImageLab, and data were plotted as the ratio of CSPα+/− to CSPα+/+ (gray bars) and CSPα−/− to CSPα+/+ (red bars). Statistical analyses were done using individual values and performed using the unpaired nonparametric Mann-Whitney U test. (C) Washed platelets (50 x 103/µL) from CSPα+/+, CSPα+/−, and CSPα−/− were resting and incubated with fluorescein isothiocyanate–conjugated anti-GPVI antibodies for 20 minutes at 37°C. Fluorescent intensities were measured by flow cytometry. Shown are representative data and geometric mean fluorescent intensity (GMFI) (mean ± standard error) of 5 independent experiments. Statistical analyses were performed using the Kruskal-Wallis multiple comparison test and corrected using the Dunn multiple comparison test. Significance: ns = P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. All error bars represent the standard error of the mean. AU, arbitrary units; HET, heterozygous; KO, knockout; ns, not significant; RabGDI, rab guanosine diphosphate dissociation inhibitor; VMAT2, vesicle monoamine transporter 2.

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