Fig. 5

Inhibition of VMP1 palmitoylation abolished the interaction between VMP1 and ALIX. A SCs were transfected with WT-VMP1-flag, MT-VMP1-flag or flag-tagged control plasmid (NC), and then conducted immunoprecipitation with anti-Flag. Total lysates and flag immunoprecipitates were then blotted. B Venn diagram of mass spectrometry results showed proteins that interacted with Flag tagged WT-VMP1, MT-VMP1, NC vectors. C Double-immunofluorescence of endogenous VMP1 and ALIX expression in SCs. The co-localization area is magnified in white box. D SCs were transfected with His-ALIX and WT-VMP1 or MT-VMP1, and tested for the interaction of exogenous VMP1 and ALIX by immunoprecipitation with anti-His antibody. β-actin served as a loading control. E The western blot of exosomal marker protein CD63, ALIX, TSG101 in sEVs obtained from SCs transfected with WT-VMP1 or MT-VMP1 and siALIX or siNC vector. F Quantification of relative exosomal protein CD63, ALIX, TSG101 in E of three independent experiments. Data were presented as mean ± SEM, *P < 0.05. ns: not significant. G Double immunofluorescence of CHMP4 and ALIX expression in SCs transfected with WT-VMP1 or MT-VMP1 vector. Right panel: Statistical analysis of the co-localization rate between ALIX and CHMP4 in the two groups. The experiment was repeated three times