Fig. 3

The CHIP mutations promote α-synuclein aggregation in SH-SY5Y and BE2-M17 cells. SH-SY5Y and BE2-M17 cells were transfected for 48 h with WT or mutant CHIP. A Confocal Images of SH-SY5Y and BE2-M17 cells following CHIP WT, p.Glu278fs, or CHIP Δ278-303 ectopic expression were captured. Scale bar, 10 μm. B, C Quantified results in A are shown as the percentage of cells with α-synuclein aggregated foci. D Ectopic expression of CHIP WT, p.Glu278fs, or Δ278–303 in A was examined using Western blot analysis. E CHIP mutants increase SDS-insoluble aggregation of α-synuclein in SH-SY5Y and BE2-M17 cells. α-synuclein aggregation was detected by the filter-trap assay in cells transfected with the CHIP WT, p.Glu278fs, or Δ278–303 plasmid. The lysate was diluted in SDS and filtered through nitrocellulose membranes. α-synuclein immunostaining was detected using α-synuclein antibody. A representative image and the densitometry data are shown (a.u., arbitrary unit). The values of α-synuclein aggregation were normalized to the amount of aggregation in the empty vector control (one-way ANOVA, *p < 0.05, ***p < 0.001). F, G The α-synuclein aggregations detected by a filter trap assay in F SH-SY5Y and BE2-M17 cells overexpressing both wild-type and mutant CHIP at the same time G were comparable between cells expressing CHIP p.Glu278fs mutant alone and those co-expressing both CHIP WT and p.Glu278fs mutant