STAT3 phosphorylation is abrogated during apoptosis. (A) Ba/F3-gp130/IL6R cells were washed 3 times and incubated for 0 hour, 2 hours, 4 hours, and 6 hours with 500 ng/mL doxorubicin in serum-free medium in the absence of IL6. The metalloprotease inhibitor marimastat was added as indicated to prevent IL6R shedding at a concentration of 10 μM. After the incubation period, the cells were centrifuged and resuspended in medium containing IL6 (200 ng/mL) or without IL6 as control for 10 minutes. The cells were immediately lysed and the proteins were immunoblotted with an anti–p-STAT3 antibody. Reprobing with an anti-STAT3–specific antibody ensured comparable protein loading. The relative IL6R cell surface expression was analyzed by flow cytometry (B) Supernatants of viable, αFas (CH-11)–treated and αFas + zVAD-fmk–treated HepG2 cells expressing the hIL6R were supplemented with 200 ng/mL IL6. After an incubation period of 30 minutes, the supernatants were used for stimulation of Ba/F3-gp130 cells for 10 minutes. Detection of p-STAT3/STAT3 was performed as described in panel A.