Figure 2
Analysis of the subcellular and cellular localization of STX-1a in PBMCs of healthy donors. (a) IF analysis of the cellular distribution of STX-1a in PBMCs. PBMCs have been immunostained with the cytoplasmic marker β-actin (upper panel) or the Golgi apparatus marker GM130 (lower panel) (green channels) and STX-1a (red channels). In the merge images, a clear colocalization of STX-1a with β-actin is evident thus indicating a cytoplasmic localization of the SNARE protein in PBMCs. A partial colocalization of STX-1a and GM130 is also shown. Original magnification: 60×. Bars correspond to 10 µm. (b) Colocalization analysis. Histogram showing the percentage of colocalized area compared to the total fluorescent area. (c) Pearson’s Coefficient. Histogram showing Pearson’s Coefficient. Green points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to β-actin, orange points and histograms represents the % of colocalization area (b) and the Pearson’s Coefficient (c) of STX-1a compared to GM130. (d) PBMC’s subcellular fractions analysis. WB analysis confirmed the cytoplasmic localization of STX-1a in PBMCs. 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 only in the nuclear fraction and that of β-actin only in the cytoplasmic one. (e) STX-1a is expressed in both major PBMCs subtypes in cultures. WB analysis reveals that STX-1a is expressed in both monocytes (M) and lymphocytes (L) and that its expression is maintained over time in cultured cells. For IF analysis at least 15 cells for each subject (n = 5) have been analyzed. For WB analysis 4 µL of PBMCs subcellular fractions (i.e. Nc and Cyto), 100 µg of whole PBMCs, 0.5 µg of mouse brain cortex lysate and 2 × 105 of cultured PBMCs have been loaded in each lane. Bars represent the median value ± SD. Uncropped WB in (d) and (e) have been reported in Fig. S5 online.
