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. 2012 Jun 12;7(6):e38545. doi: 10.1371/journal.pone.0038545

Figure 1. CL1 sequesters α-syn to the detergent-insoluble fraction of SHSY5Y cells.

Figure 1

(A) SHSY5Y cells expressing WT, E46K, A53T, WT-CL1, E46K-CL1 or A53T-CL1 α-syn were fractionated to soluble and insoluble fractions and analyzed by Western blot. Attachment of CL1 to WT or mutant α-syn reduced their solubility in the SHSY5Y cells. (B) SHSY5Y cells expressing WT or WT-CL1 α-syn were subjected to cycloheximide pulse chase analysis. Cells were harvested at different time points and subjected to Western blot analysis. The degradation of WT-CL1 α-syn was enhanced when compared to WT α-syn. (C) Three independent cycloheximide pulse chase experiments were performed and the levels of WT or WT-CL1 α-syn were quantified using optical densitometry (*p<0.05; **p<0.01). (D) GST, GST-WT α-syn or GST-WT-CL1 α-syn was mixed with or without liposome and subjected to lipid sedimentation assay. The samples were then analyzed by SDS-PAGE with Coomassie stain. Attachment of CL1 to α-syn did not affect its lipid binding affinity.