Research Paper Volume 12, Issue 20 pp 20702—20727

BAG5 expression is crucial for α-synuclein aggregation. (A) After rotenone treatment (10 μM) for 24 h, images of rotenone-treated α-synuclein (A53T)-expressing SH-SY5Y cells following BAG5 knockdown were captured. Arrows indicate α-synuclein-containing foci. Scale bar, 10 μm. (B) Quantified results in (A) are shown as the percentage of cells with foci (Student’s t-test; **, p < 0.01). (C) Knockdown of BAG5 in (A) was displayed by Western blot analysis. (D) BAG5 increases SDS-insoluble aggregation of α-synuclein in U2OS, HeLa, and SH-SY5Y cells. α-Synuclein aggregation was detected by the filter-trap assay in cells transfected with various amounts of a BAG5-expressing plasmid. The lysate was diluted in SDS and filtered through nitrocellulose membranes. α-Synuclein immunostaining was detected by the 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 (Student’s t-test; *, p < 0.05). (E) SDS-insoluble α-synuclein aggregation in rotenone-treated and/or α-synuclein (A53T)-expressed SH-SY5Y cells is BAG5-dependent. α-Synuclein aggregation was detected by the filter-trap assay. The values of α-synuclein aggregation were normalized to the amount of aggregation in the vector or solvent control (Student’s t-test; *, p < 0.05).