FIGURE 7.

The disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A, HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody (green), an anti-FLAG antibody (red) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B, HeLa cells were transfected and stained as in panel A except an anti-calnexin antibody was used instead of WGA to define the limits of the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10 or HA-ADAM10 17DCS signal localized in the ER. Data are representative of three independent experiments and at least 15 fields of view. A two-way ANOVA statistical analysis was performed with a Bonferroni's multiple comparisons test (ns, non-significant, ****, p < 0.0001). C, HEK-293T cells were mock transfected (−), or transfected with HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HA antibody. Immunoprecipitates were stained with neutravidin (top panel) or an anti-HA antibody (bottom panel). Whole cell lysates were stained with an anti-HA antibody (middle panel).