Reduced levels of RAX prevent IFNγ/TNFα-induced apoptosis. (A) IFNγ up-regulates PKR but not RAX in MEF cell lines. Western blots using antibodies to PKR or RAX with lysates from cells treated with 10 ng/mL IFNγ and TNFα. (B) Viability of MEF cells expressing either exogenous RAX or RAX(S18A) compared with vector control cells after treatment with 10 ng/mL IFNγ and/or 10 ng/mL TNFα for 24 hours. (C) Viability of MEFs expressing RAX, RAX(S18A), or reduced RAX that were pretreated with 20 ng/mL IFNγ for 24 hours followed by increasing concentrations of TNFα for 24 hours measured by TUNEL assay. (D) Relative PKR activity was assessed by autophosphorylation assay. Results represent the averaging of 3 separate experiments that quantify activated PKR and total PKR by autoradiography and Western blotting. Cells were treated with 10 ng/mL IFNγ for 24 hours followed by addition of 10 ng/mL TNFα for the indicated times. (E) Relative eIF2α phosphorylation determined by quantifying Western blots from 3 separate experiments using antibody to eIF2α and phosphoserine-51 eIF2α following treatment with 10 ng/mL IFNγ for 24 hours and with 10 ng/mL TNFα for the indicated times.