Figure 8. Analysis of Tspan5/Tspan15 chimeras.

(A) Schematic representation of the various chimera used. (B) Analysis using the confocal microscopy protocol of ADAM10 endocytosis in HeLa cells transfected with Tspan5, Tspan15, or the different chimeras. Data are expressed as a function of the endocytosis rate measured in parental HeLa cells. (C) Analysis of Notch signaling in U2OS cells transfected with the different chimeras and stimulated with OP9 cells expressing the Notch ligand DLL1. The data are expressed as a percentage of the signal observed for control U2OS cells. (D) Relative ADAM10 expression in U2OS cells expressing the various chimeras, determined by flow-cytometry. (E) U2OS cells transfected or not with Tspan5 or Tspan15, or the chimeras T5C15 and T15C5 were incubated for the indicated time with 10 μg/ml DyLight 650–labelled anti-ADAM10 mAb. After detachment, the surface pool of ADAM10 was stained using a PE-labelled secondary reagent before flow-cytometry analysis. The graph shows the evolution in time, and relative to cells stained at 4°C after detachment, of DyLight 650 fluorescence. This figure summarizes the data obtained in different experiments in which HeLa cells and Tspan5- and Tspan15-transfected cells were always analyzed as a reference. In (E), data were statistically analyzed after normalization using a one-way ANOVA followed by a Dunnett’s multiple comparisons test. In the other panels, data were log-transformed before statistical analysis on the various sets of experiments using a repeated measures one-way ANOVA. Each transfectant was compared with parental cells using the Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.