FIGURE 3

FasL cleavage is mediated by ADAM10 in fibroblasts. MEFs were transfected with hFasL. After 6 h, the culture medium was removed and the cells were cultured for 24 h before cells and supernatants were analyzed by immunoblot. For ELISA-analysis, sFasL generation over 12 h was measured. (a) Supernatants and pellets of the indicated MEFs were subjected to ELISA for detection of FasL. To normalize variations in transfection efficiency for each cell line sFasL generation was calculated as ratio of sFasL in the supernatant to FasL in cell pellets (CP-FasL). Data of three independent experiments are expressed as meansstandard error of the mean (S.E.M.). (b) Release of sFasL in the medium of untransfected and FasL-transfected wild-type MEFs and ADAM10^-/- cells was analyzed by western blotting. Despite comparable amounts of full-length FasL (middle panel), sFasL was only detected in supernatants of FasL-transfected wild-type MEFs (upper panel). The expression of ADAM10 in the same cell lysates is shown in the lower panel. (c) FasL-transfected ADAM10^-/- MEFs were retransfected with ADAM10 (ADAM10^-/-/ADAM10-transfected) or ADAM17 (ADAM10^-/-/ADAM17-transfected) and compared with wild type and ADAM10^-/- MEFs for the production of sFasL by ELISA. Data of three independent experiments are expressed as meansstandard error of the mean (S.E.M.). (d) ADAM10^-/- MEFs were retransfected with ADAM10 (ADAM10^-/-/ADAM10-transfected) and compared to wild type and ADAM10^-/- and ADAM10^-/-/ADAM17-transfected cells for the generation of the FasL NTFs. Western blots are shown for FasL (Ab3), HA-tagged ADAM10, HA-tagged ADAM17 and -actin. NTF, NTFs of FasL; FL, full-length FasL