Figure 2: ARF6 and ARF6 cycling are required for syntenin exosome release.

MCF-7 cells were transiently transfected with an expression vector for wild-type ARF6 (+ARF6-WT), an expression vector for the fast cycling mutant of ARF6 (+ARF6-T157N) or the empty vector (pCDNA3) as control. (a) Total cell lysates and corresponding exosomes were collected 24 h after transfection and analysed by western blotting for different markers, as indicated. (b) Histograms represent mean±s.d. signal intensities in exosomes, relative to signals in siCNT exosomes, calculated for n independent experiments. (c) MCF-7 cells were treated with ARF6 RNAi (siARF6#2 and siARF6#1) or non-targeting RNAi (siCNT) for 48 h and then transiently transfected with an expression vector for wild-type syntenin (+ Syntenin) or the empty vector (pCDNA3) as control. Total cell lysates (c) and corresponding exosomes (d) were collected 24 h after transfection and analysed by western blotting for different markers, as indicated. (d) Histograms represent mean signal intensities±s.d. in exosomes, relative to signals in siCNT exosomes, calculated for n independent experiments. siARF6#1 and siARF6#2 data were pooled for quantification and statistical analysis. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).