Figure 3.

(See previous page). Characterization of particle fractions (PFs) prepared from conditioned medium of transfected H4 cells. (A) to (F) Western blot and dot blot analysis of SNCA associated with PFs prepared from conditioned medium of H4 cells expressing high-aggregating SNCA-T and SNCAIP or low-aggregating SNCA. (A) Representative protein gel blots of total SNCA⁺ PFs prepared from 12 ml conditioned medium of transfected H4 cells -/+BafA1 show an increased presence of extracellular SNCA (14 kDa) or SNCA-T (27 kDa) after BafA1 treatment. (B) 12 ml supernatant after PF separation contain only low levels of soluble SNCA. (C) Representative western blots of SNCA PFs -/+BafA1 after filter retardation to exclude particles of different size by using distinct pore size filters reveal enlarged particles (>1 .2 μm) in the presence of BafA1. (D) Representative dot blots of SNCA in the conditioned medium of H4 cells expressing low-aggregating SNCA -/+BafA1. (E) Representative protein gel blots of SNCA-T and SNCAIP PFs after exclusion of particles of different size by using distinct pore size filters. (F) Quantification of SNCA associated with PFs after using filters of defined pore size normalized to SNCA levels in unfiltered PFs displaying a shift toward smaller particles by BafA1. (G) BafA1 effect on oligomerization analysis of SNCA associated with PFs prepared from H4 cells expressing SNCA, as well as SNCA-T and SNCAIP measured by sucrose gradient centrifugation. All values are mean + s.e.m. Differences are significant at (*) P < 0.05.