Fig. 3. Proteasome inhibition triggers TACE-dependent release of TNF- without altering the amount of TACE antigen. (A) ECV-304 cells stably expressing TNF- were incubated for 20 minutes without (white bars) or with 300 nM of TIMP-3 (black bars) or TIMP-1 (striped bars) before MG-262 (100 nM) was added or not. TNF- that had accumulated in the culture medium over a period of 4 hours was measured by ELISA. (B) ECV-304 cells were co-transfected with TNF- and a dominant-negative form of TACE (DN), or a control vector (Cont). Fourty-eight hours after the transfection, cells were treated or not with MG-262 as described for Fig. 2. TNF- that had accumulated in the medium over a period of 4 hours (black bars) and was present in the cell lysates at the end of the experiment (white bars) was measured by ELISA. (C) The total cellular amount of TACE was immunoblotted as described in Materials and Methods. The immature form of TACE is indicated by `i', the mature form by `m'. (D) The cell surface expression of TACE was measured by flow cytometry. Experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.