Fig. 5. The molecular mass of T735E.TACE corresponds to mature TACE and is processed to maturity quicker than pro-WT.TACE. (A) COS-7 cells were transfected with 3 µg pcDNA3.1+ (lane U) or 3 µg pWT.TACE (lane NIgG and WT.TACE), pT735A.TACE (lane T735A.TACE) and pT735E.TACE (lane T735E.TACE). Cells transfected with pT735E.TACE were treated with 50 µM leupeptin for 18 hours after 6 hours of transfection. Twenty-four hours after transfection, the cells were lysed and TACE was immunoprecipitated using non-immune IgG antibodies (lane NIgG) or the anti-TACE ICD specific antibody. Immune complexes were washed with NP-40LB, and analysed by western blotting with the anti-TACE ICD antibody (C-15). (B) Similarly, 2x10⁶ WT.HeLa or untransfected HeLa cells were seeded overnight and TACE immunoprecipitated using the anti-TACE antibody (C-15). After extensively washing the immune complexes using NP-40LB they were analysed by western blotting using an anti-TACE antibody (C-15). (C) COS-7 cells were transfected with pWT.TACE, pT735A.TACE and pT735E.TACE and treated with 50 µM leupeptin for 18 hours after 6 hours of transfection. Cells were radiolabelled for 3 hours with [³⁵S]methionine/cysteine in the presence of 50 µM leupeptin, washed and chased in complete medium (lacking leupeptin) for the time indicated. TACE was purified and analysed by SDS-PAGE and autoradiography.