Fig. 4

A53E does not change the inclusion pattern. a-b At least 50 cells per condition were classified according to the pattern formed. We observed that A53E did not change the number of inclusions per cells. n = 3. Scale bar: 30 μm. c-e Immunoblot analysis of the aSyn and Synphilin-1 levels showed no significant differences in expression of WT or A53E aSyn. n = 3 (f) Inclusions formed by WT and A53E are positive for pS129. Positive inclusions are indicated with white arrows. Scale bar: 30 μm. g Inclusions were stained with Th-S and analyzed via fluorescence microscopy. As indicated with arrow heads, we observed that some inclusions displayed amyloid-like properties, by staining positive with Th-S. Scale bar: 30 μm. h-i WT and A53E aSyn protein lysates were digested with PD for different times (1, 3 and 5 min). After normalization of the values to the undigested condition, we observed that A53E inclusions are less resistant to PK-digestion. n = 2. j-k 48 h post-transfection the cells were collected and we assessed the activity of the proteasome. We observed that cells expressing A53E mutant increased proteolytic activity of the proteasome in comparison with WT. n = 3. Student’s t test (*p < 0.05, **p < 0.01)