Fig. 6

The C-terminal cytoplasmic region mediates the interaction of TSPAN6 with syntenin-1. a Table illustrating the location of the point mutations on the myc-tagged TSPAN6 mutants used in the co-immunoprecipitation experiment shown in b. b Co-expression of Flag-tagged syntenin in HEK293 cells together with myc-tagged TSPAN6-wt or lacking one of the last c-terminal amino acids (TSPAN6(-1) to TSPAN6(-7)). After immunoprecipitation of Flag-tagged syntenin, the interaction with the different TSPAN6 mutants was evaluated by western blot with an anti-myc antibody. The last 2 amino acids of TSPAN6 are essential for the interaction with syntenin. c N-terminally biotinylated peptides corresponding to the indicated C-termini of tetraspanins were synthesized and conjugated to avidin-agarose beads. HEK293 cells ectopically expressing HA-tagged syntenin were lysed in immunoprecipitation buffer (0.5% Brij 98-0.5% Triton X-100-PBS), incubated with the avidin-immobilized peptides and the interaction to syntenin was analyzed by western blot. TSPAN6 (Tsp6) and CD63 (positive control) C-terminal peptides also interacted with syntenin. Net-5, Net 2 (negative controls reported not to interact to syntenin), CD231 or CD81 did not interact to syntenin