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Fig. 2. In ECV-304 cells, the ectodomain-shedding of ectopically expressed TNF- is under the control of a TACE-like activity and is stimulated by proteasome inhibition. (A) Ro 32-7315 (5 µM) was added or not to ECV-304 cells stably expressing TNF-, 15 minutes before the cells were incubated for 4 hours with PMA (20 nM) or MG-262 (600 nM) as indicated. TNF- that had accumulated in the medium (black bars) and was present in the cell lysates at the end of the experiment (white bars) was measured by ELISA. Inset shows the mRNA levels of TNF- and eukaryotic elongation factor (EF1) analyzed by RT-PCR in (lane 1) ECV-304 cells and (lanes 2-4) ECV-304 cells expressing TNF- under basal conditions (lanes 1, 2), and after 4 hours of (lane 3) MG-262 or (lane 4) PMA treatment. (B) ECV-304 cells stably expressing TNF- were treated for 4 hours with MG-262 (600 nM), lactacystin (40 µM) or E-64d (10 µM), and TNF- that had accumulated in the medium was measured by ELISA. Cont: control; Lac: lactacystin; Ro: Ro 32-7315. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.

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