Intrinsic induction of apoptosis induces ADAM17-mediated IL6R shedding. (A) Ba/F3-gp130/IL6R cells were treated for 2 hours, 4 hours, and 8 hours with doxorubicin (500 ng/mL) or 2 hours with the phorbol ester PMA (100 nM). Soluble IL6R was immunoprecipitated from conditioned media using the polyclonal anti–serum 6.2²⁶  and subsequently visualized by Western blot analysis using the anti-IL6R mAb 14-18. Reciprocal Western blot analysis of cell lysates was performed using mAb 14-18 and an anti–β-actin mAb, which was used as a loading control. (B) Ba/F3-gp130/IL6R cells were treated for 6 hours with doxorubicin, and multiparameter FACS staining was performed with propidium iodide and antibodies against annexin V, and the anti-IL6R (M91 mAb). Histograph assignments are as follows: (1) viable cells, (2) early apoptotic cells, and (3) late apoptotic cells. (C) Ba/F3-gp130/IL6R cells were treated for 8 hours with 500 ng/mL doxorubicin or for 2 hours with 100 nM PMA in the presence or absence of the metalloprotease inhibitors GW280264X (3 μM) and GI254023X (3 μM). Inhibitors were added 30 minutes prior to stimulation. Western blot analysis was used as outlined for panel A to monitor levels of sIL6R, IL6R, and β-actin. (D) Ba/F3-gp130/IL6R cells were treated as described in panel C and multiparameter FACS analysis was performed as before. The intensity of IL6R staining was monitored in both viable (gray histograms) and apoptotic (white histograms) cells in the presence or absence of the indicated inhibitors.