Fig. 4. Proteasome inhibition specifically stimulates TACE-dependent ectodomain shedding. (A) ECV-304 cells were transiently transfected with TNFR2. Fourty-eight hours post transfection cells were treated as described for Fig. 2. Ectopically expressed TNFR2 (black bars) and endogenous TNFR1 from untransfected cells (white bars) that had accumulated in the medium over a period of 4 hours were measured by ELISA. (B) Cells ectopically expressing TNFR2 at their surface were counted by flow cytometry. (C) Endogenous ICAM-1, released in the culture media of ECV-304 cells, was measured by ELISA and its cell surface expression was measured by flow cytometry. (D) Epifluorescence microscopy of the intracellular distribution of ICAM-1 in untreated and MG-262-treated ECV-304 cells. Flowcytometry experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.