Fig. 3: Retromer-mediated cargo transport is regulated by Hsc70 activity-dependent binding and dissociation of DNAJC13 from SNX1.

A Co-IP analysis of GFP-DNAJC13^wt and FLAG-SNX1 in COS7 cells exposed to different concentrations of the Hsc70 inhibitor VER-155008 for 24 h. Treatment with VER-155008 markedly increased the binding of DNAJC13 and SNX1 in a dose-dependent manner. B Confocal images of the COS7 cells exposed to VER-155008 co-immunostained with M6PR (green) and TGN46 (magenta). The VER-155008 treated cells show more dispersed punctate distribution of M6PR compared to the untreated cells. The white dotted lines indicate outline of the cells. The inset shows a magnified image of the white square. Scale bar: 20 μm. C Co-localization ratio of M6PR and TGN46 in VER-155008-treated cells is significantly less than that of the untreated cells. Data were statistically analyzed using the Kruskal–Wallis and post hoc Bonferroni tests. *p < 0.05, n = 31 (0 μM), 30 (10 μM), and 28 (25 μM). D Co-IP analysis of GFP-DNAJC13^wt and FLAG-SNX1 in COS7 cells in the presence or absence of rotenone treatment at a final concentration of 0.1 μM for 3 days. Binding of SNX1 to DNAJC13 was enhanced with rotenone treatment. E Confocal images of the rotenone-treated cells co-immunostained with M6PR (green) and TGN46 (magenta). The white dotted lines indicate the cell outlines. The inset shows a magnified image of the white square. Scale bar: 20 μm. F Co-localization ratio of M6PR and TGN46 was lower in the rotenone-treated cells than in the untreated cells. Data were statistically analyzed by Mann–Whitney U test. *p < 0.05, n = 36 (untreated group) and 36 (rotenone-treated group).