Cytokine deprivation of Ba/F3 cells induced apoptosis and IL6R shedding. (A) IL3-dependent Ba/F3-gp130/IL6R cells were washed 3 times and cultured for 12 hours and 24 hours in cytokine-free medium to induce apoptosis. Control cell cultures were maintained over the 24-hour period in the presence of IL3 (IL3WD, 0 hour; WD, withdrawal). Marimastat (10 μM) was added as indicated. Lysates and immunoprecipitated supernatants were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot with anti-IL6R antibody 14-18. (B) IL6-dependent Ba/F3-gp130/IL6R cells were treated and analyzed as described in panel A. The metalloprotease inhibitors marimastat (10 μM), GI254023X (3 μM), and GW280264X (3 μM) were added as indicated. (C) IL6-dependent Ba/F3-gp130/IL6R cells were treated as described in panel A. Cells were washed and stained with the anti-IL6R antibody M91 and analyzed by flow cytometry. The black bar represents isotype control staining, the light gray bar represents IL6R surface expression of viable cells, and the dark gray bars represent IL6-deprived cells in the presence or absence of the metalloprotease inhibitors described in panel B. Values represent the mean (± SD) from 3 independent experiments (P ≤ .01).