Figure 5

pMac degrade exogenous αS. (A) Immunocytochemistry of pMac: αS (green); lysosomal marker LAMP1 (red); nuclei (DAPI, blue; scale bar = 20 µm), region within white square is magnified below. (B) As (A) but pMac treated for 2 hrs with 10 µg/ml αS. (C) As (B), but pMac then washed and replated (4 hrs). (D) pMac incubated for 2 hrs with 10 µg/ml monomeric αS, washed and replated for the indicated number of hrs before assaying for intracellular αS by FACs; MFI relative to endogenous αS in untreated pMac. (E) Release of αS to supernatant of same experiment as (D). (F) As (D), with the addition of degradation pathway-specific drugs upon replating pMac after αS exposure and incubation for 8 hrs. (G) Pathway-specific drugs were added to cells (without exogenous αS exposure) for 2 and 8 hrs and assayed for intracellular accumulation of endogenous αS by FACs. (F,G) 1 line, 3 independent differentiations. Statistical analyses: (E) two-tailed t-test; (D,F,G) one way ANOVA with Dunnett’s multiple comparisons test.