Fig. 5
The RidL9–258 β-hairpin displaces TBC1D5 from the retromer. a HeLa cells were transiently transfected with vectors producing the GFP-fusion proteins or GFP as indicated and immuno-stained for TBC1D5 (cyan) and the retromer (Vps26; red). Scale bar, 10 μm. b Co-localization of TBC1D5 with retromer (Vps26) shown in a was scored. Pearson’s correlation coefficient was calculated in three independent experiments with at least 50 cells each (mean and SD of the means from each experiment; one way ANOVA; ***P < 0.001). c GFP-TBC1D5 and associated endogenous retromer components were pulled down from transiently transfected HeLa cell lysate. The retromer was eluted from immobilized GFP-TBC1D5 with purified RidL10–258, RidL10–258-Δβ-hairpin or no protein. Input lysate (lys, 1%), washed beads after elution (beads, 20%) and eluates (20%) were analyzed by SDS-PAGE, Western blot and immuno-staining of GFP (GFP-TBC1D5: 117 kDa), Vps35 (91 kDa), Vps26 (40 kDa) or RidL (RidL10–258: 29 kDa, RidL10–258-Δβ-hairpin: 27 kDa). The bands of retromer components were quantified relatively to the value for mock-treated beads (indicated in %). One representative experiment of three independent biological replicates is shown. m, marker. d HeLa cells were transiently transfected with vectors producing RidL9–258-GFP or GFP, and the trafficking of Shiga toxin subunit B (STxB)-Cy3 was analyzed by co-localization with the Golgi-marker GM130 after 30 min. RidL9–258-GFP did not affect trafficking of STxB-Cy3. As a positive control, GFP-producing cells were treated with 25 µM Retro-2 30 min prior to and during the trafficking assay. Images shown are representative of three independent experiments. Scale bar, 10 µm
