FIGURE 2.

The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A, schematic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable (var) region of CD9 (black) and Tspan14 (gray) were interchanged; the N-linked glycosylation site of Tspan14 is indicated by a filled oval. B, HEK-293T cells were mock transfected (−) or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (+). Cell lysates were produced using 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-Myc tag antibody (middle panel). Data are representative of three independent experiments. C, quantitation of immunoprecipitated ADAM10. Data in panel B (upper panel) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock (****, p < 0.0001). Error bars represent standard error of the mean from three experiments. D, data in panel B (middle panel) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (***, p < 0.001).