Fig. 5

Vps13B binds to Stx6- and Stx13-positive vesicles. a Recruitment of Vps13B and EEA1 to liposomes containing Stx6, Stx13, Stx6, and Stx13 together. For details, see legend to Fig. 4b. α-Tubulin was used as loading control, because the protein binds to liposomes dependent on the lipid composition (ref. ²³). b, c Co-immunoprecipitation of Vps13B and SNARE proteins; b shows co-precipitation of Stx6 and 13 but not of any other SNARE with Vps13B. c Conversely, Vps13B co-precipitates with Stx6 and Stx13 but not with Stx16 and Stx4. The bottom panel shows that all SNAREs were efficiently immunoprecipitated. d The N-terminal domain of Stx13 is necessary for binding to Vps13B. Lysates from cells expressing Vps13B-FLAG and GFP-Stx13, GFP-ΔN-Stx13, or GFP-Stx13-Stx6 chimera were immunoprecipitated with anti-FLAG antibody and probed by immunoblotting for GFP. e Immunofluorescence for Vps13B of cells expressing mRFP-Stx6 and GFP-Stx13. Arrows in the insert indicate triple-positive vesicles. Scale bar, 10 µm. f Co-localization between Stx6, Stx13, and Vps13B in the peripheral region of the imaged HeLa cells. Vesicles were counted from nine images, obtained in three independent experiments, with the degree of overlap shown in the bar graph on the right (numbers denote total number of vesicles analyzed). g Co-localization of Vps13B with TfnR, EEA1, M6PR, and Golgin97. White arrows show co-localized vesicles. Scale bar, 10 µm. Intensity plots of exemplary line scans are shown on the right. A bar graph shows the percentage of TfnR-, EEA1-, M6PR-positive vesicles, which are also positive for Vps13B