Fig. 5: CD63-WT is less efficiently released in EVs than CD9 and CD63-YA. | Nature Communications

Fig. 5: CD63-WT is less efficiently released in EVs than CD9 and CD63-YA.

From: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Fig. 5

a Western blot of the cell lysate (CL) and of the different EV pellets obtained by differential ultracentrifugation of CCM from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP or CD63-YA-mCherry (24 h release with biotin). EVs from 20 × 106 cells and CL from 0.2 × 106 cells were loaded. The intensity of the band corresponding to the mCherry fusion proteins was quantified and normalized by the intensity of the CD9 band in 3 independent experiments, the mean ± SD is represented. Two-tailed paired t test. b Representative Western blot of the pull-down (PD) and flow-through (FT) of the immunoprecipitation of EVs from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP, CD63-YA-eGFP, or CD9-eGFP, recovered 24 h after biotin addition. 60 × 108 total particles quantified by NTA were used for each IP. Percent of GFP + cells quantified by flow cytometry were similar in the three conditions (Supplementary Fig. 5a). The GFP bands intensity in the PD normalized to endogenous CD9 in the corresponding PD are represented as mean ± SD for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. c Representative Western blot of EVs (200 K pellets) from HeLa cells transfected with the CD63-WT, CD63-YA, or CD9-eGFP RUSH plasmids treated with DMSO of BafA1 100 nM during 16 h. The same number of EVs between the DMSO and the BafA1 conditions were loaded on the gel (around 100 × 108 particles). The fold change between DMSO and BafA1 treatment for each construct is represented as mean ± SD for 3 independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 1. d Proportion of cellular endogenous CD9 and CD63 released in EVs, as semi-quantified on Western blots. The signal for CD9 and CD63 in 200 K pellets released by 20 × 106 HeLa cells was divided by the signal for the same molecule in the total lysate of 0.2 × 106 cells, run on the same blot. 1 representative Western blot and quantification (mean ± SD) of 3 independent experiments. Two-tailed paired t test.

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