Figure 2. GGA3 enhanced α-syn oligomer secretion is mediated mainly by exosome-free pathways. Conditioned media from N2A cells co-expressing S1/S2 and GGA3 or myc control were collected 48h after transfection. Exosomes were isolated from CM by subcellular fractionation. Purity of the fractions was confirmed by Western blot and the exosome-specific marker flotilin (A). As previously observed, α-syn oligomers, as measured by the luciferase activity, were increased in the conditioned media (A, lane4+8) but not cells (A, lane3+7) of GGA3 over-expressing cells compared to control. α-syn oligomers were detectable in exosomal fractions of both GGA3 over-expressing cells and controls with a small increase in exosomes (A, lane1+5) in the GGA3 condition but there was a higher increase in exosome depleted medium (A, lane2+6). Measurements were carried out in triplicate and the mean counts per second/100µl ±SEM of one representative experiment are shown. Normalization to myc control revealed a ~3-fold increase of α-syn oligomers, as measured by the luciferase activity, in the exosome-free supernatant of GGA3 over-expressing cells (B, lane2+6) and only a ~1.5- fold increase in the exosomal fraction (B, lane1+5). The mean fold change over the control ±SD of n=3 independent experiments is shown. Statistical analysis was performed by t-test with *=p<0.05, **=p<0.01,***=p<0.001.