Figure 1.

α-Synuclein is constitutively released by a nonclassical secretory pathway. A, SH-SY5Y cells were cultured in the presence or absence of Dox for 8 d in 10% FBS. After that period, the culture medium was replaced with 2% FBS medium for the indicated times. Cell lysates (CL; left) and concentrated CM (right) were analyzed by Western immunoblotting with antibodies to α-synuclein or ubiquitin (Ubi). ERK and BSA levels were used as loading controls. B, SH-SY5Y cells were cultured as in A, and the CM was collected after 48 h and analyzed by Western immunoblotting with C-20 antibody. C, Representative plot showing the levels of secreted α-synuclein, estimated as the percentage ratio of the extracellular α-synuclein versus the intracellular α-synuclein, over time. Each point represents the mean ± SD of three measurements. D, α-Synuclein-expressing cells were treated with 2 μg/ml BFA (+) or without (−) for 6 h. The levels of intracellular α-synuclein (CL) and extracellular α-synuclein (CM) were assessed by Western immunoblotting. ERK and BSA were used as loading controls.