Figure 5
Confirmation of increased STX-1a levels in PBMCs of IS patients with respect to HC subjects. (a) Representative WB of STX-1a expression levels in PBMC cytoplasmic fractions. Representative WB analysis of cytoplasmic and nuclear fractions of PBMCs. For each sample, the STX-1a densitometric values have been normalized to that of β-actin. The successful separation of the nuclear (Nc) and the cytoplasmic fractions (Cyto) of PBMCs is demonstrated by the presence of the nuclear marker lamin A/C in the nuclear fraction and that of β-actin in the cytoplasmic one. (b) Quantitative analysis of cytoplasmic STX-1a. STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a significantly increase in PBMCs of IS patients with respect to HC (p ≤ 0.0001) and Y-HC (p ≤ 0.003) subjects, while no difference is observed between the two control groups (p = 0.7). (c) Representative Immunofluorescence analysis of STX-1a in PBMCs of IS patients and HC subjects. PBMCs have been labeled with β-actin (green) and STX-1a (red). The STX-1a fluorescence intensity has been normalized by dividing the measure of its intensity by that of the β-actin subtracted from that measured for the nucleus (DAPI immunostaining). (d) Quantitative analysis STX-1a levels in PBMCs. The normalized STX-1a expression levels have been analyzed by using One-way ANOVA with post-hoc Tukey test: STX-1a levels are significantly increased in PBMCs of IS patients with respect to HC subjects (p = 0.005). Original magnification: 60X. Bars correspond to 10 µm. For WB analysis 4 µL of each fraction (Nc and Cyto) and 100 µg of whole PBMCs have been loaded in each lane. For IF analysis at least 15 cells for each subject have been analyzed. In quantitative graphs (panels b and d) each point in a frame depicts the value for a single subject (n = 5–10 IS patients, 5–10 HC subjects and 0–5 young HC subjects, respectively in (b) and (d) while bars represent the median value ± SD. **p ≤ 0.005; ***p ≤ 0.001. Uncropped WB in a) have been reported in Fig. S8 online.
