Fig. 6: Proteomics pathway analyses of VPS35-interacted proteins and VPS35-HSC70 interaction in α-Syn aggregation.

a Pathway analyses of the proteomics dataset of VPS35 immunoprecipitants (IP) from mouse retinal lysates. Selected enriched pathways of VPS35-interacting proteins analyzed by DAVID are shown in (a). The -Log₁₀ of P-values from one-sided Fisher’s exact test are shown. b, c The enriched Gene Ontology (GO) pathways were identified by one-sided Fisher’s test and the GSEA method. The P-values from the Fisher’s test were adjusted by using Bonferroni correction. There are 63 significant GO pathways with adjusted P-value < 0.05 from Fisher’s test and 41 significant GO pathways with FDR < 0.2 from GSEA. Twelve pathways were shared by the two significant GO pathway sets obtained from Fisher’s test and GSEA. For these pathways, the -Log₁₀ of P-values from Fisher’s test are shown in (b) and the normalized enrichment scores from GSEA are plotted in (c). d Pull-down assays. GST alone or GST-VPS35 fusions conjugated on glutathione beads were mixed with HSC70. The glutathione elutes were immunoblotted (IB) with GST and HSC70. The expected size of GST fusions of VPS35 full-length (FL), C-terminal fragment (C), and N-terminal fragment (N) are shown. The relative ratio of the signal intensity of HSC70: GST (GST alone of GST-VPS35 fusions), considering GST alone as 1, are shown. e, f Immunostaining of HSC70 together with VPS35 (e) or EEA1 or Lamp1 (f) in 661 W cells is shown. The overlapped signals are highlighted by arrows in insets. g–i 661W cells expressed plasmids encoding Rab5Q79L, Myc-αSyn, and pRK5 (Ctrl) or VPS35-shRNAs (KD) for 3 days and stained for indicated antibodies. g Representative images show VPS35-KD cells expressed prominent HSC70⁺/αSyn⁺ aggregates in CD63-labeled LEs (arrows). h Quantification shows significantly more VPS35-KD cells exhibited αSyn⁺/HSC70⁺ aggregates in Lamp1-labeled LEs. More than 200 transfected cells per condition were counted in 3 independent assays. Mean ± SD and P-values. Two-way Student’s t-test. i VPS35-KD cells expressed prominent Myc-αSyn⁺, P-αSyn⁺ aggregates in Lamp1+ LEs (arrows). Ctrl cells expressed diffused Myc-αSyn and P-αSyn signals instead. Representative images of 3 independent images of (d–i) are shown. Source data of (h) provided as a Source Data file.