Fig. 7

The interaction of syntenin with TSPAN6 mediates the enhanced formation of exosomes. a Flag-tagged TSPAN6 (Flag-TSPAN6) and HA-tagged syntenin (SynWt-HA) or syntenin lacking either the N-terminal fragment (SynΔN-HA) or the C-terminal fragment (SynΔC-HA) were co-transfected in HEK293 cells. Flag-TSPAN6 was immunoprecipitated with an anti-Flag antibody from cell lysates and the interaction was evaluated by western blot using an anti-HA antibody. Total lysates (first 3 lanes) show that the 3 syntenin constructs were expressed. However only SynWt-HA and SynΔN-HA but not SynΔC-HA were immunoprecipitated with Flag-TSPAN6. The absence of anti-Flag antibody gives no bands (negative control, last 3 lanes). b Nuclear magnetic resonance spectroscopy (NMR) analysis of syntenin-1 and TSPAN6 interaction. The left panel shows the superposition of six two-dimensional 1H-15 N-HSQC spectra of uniformly 15 N-labeled syntenin-1 PDZ domain (200 μM) titrated with increasing concentrations of C-terminal peptide of TSPAN6. The right panel shows the locations of syntenin PDZ residues involved in binding. c Exosomes isolated from the conditioned medium of HEK293 cells stably knocked down for syntenin (HEK-Syn KD) and overexpressing an empty vector (control) or TSPAN6 were analyzed by Nanosight. Overall, the number of exosomes is lower in HEK-Syn KD cells compared to HEK293 wt cells (Fig. 5f ). Overexpression of TSPAN6 in HEK-Syn KD did not alter the number of exosomes secreted. d Same plot as in Fig. 5  g but for HEK293 cells stably knocked down for syntenin (HEK293-KD Syntenin). Neither number of exosomes nor size distribution change between TSPAN6-overexpressing and control cells (data from panel c). e HEK293 or HEK-Syn KD were co-transfected with an empty vector (control) or with TSPAN6 (n = 3 technical replicates). TSPAN6 overexpression increases the APP-CTF levels in both HEK293 and HEK-Syn KD cells. f Quantification of the western blots of panel e. g HEK293 cells were transfected with an empty vector (control), TSPAN6 alone or with either syntenin (Syn) or syntenin with a point mutation in PDZ1 domain (PDZ1) that prevents the interaction to TSPAN6. Lysates were analyzed by western blot (n = 3 technical replicates). Overexpression of TSPAN6 increases the APP-CTF levels. This effect is potentiated by co-expression of Syn or PDZ1 mutant. h Quantification of APP-CTF levels from the western blot shown in panel g. Statistical analysis was carried out with t-test (** p <0.01 vs vector; ## p0.01;vs TSPAN6 alone)