FIGURE 6.

All TspanC8s interact with the region of ADAM10 comprising the disintegrin (D), cysteine-rich domain (C), and stalk (S). A, HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or negative control (−). Lysates were extracted in 1% digitonin lysis buffer and proteins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). B, data in panel A (upper panel) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Data are 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. All TspanC8s bound significantly to ADAM17 10DCS (p < 0.0001). Error bars represent standard error of the mean from three experiments. C, ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbitrarily set at 100. Error bars represent standard error of the mean from three experiments.