Anti-Fas MoAb induces activation of SAPK and p38 MAP kinase. (A) RPMI 8226 MM cells were treated with 5 μg/mL of anti-Fas MoAb for 15, 30, and 60 minutes. Cell lysates prepared from control cells (media alone) and anti-Fas MoAb-treated cells were incubated with glutathione-sepharose beads containing 5 μg of GST-Jun (2-100) for 2 hours. The resulting protein complexes were washed and then incubated in kinase buffer containing GST-Jun fusion protein and [γ-³²P]ATP for 15 minutes at 30°C. The phosphorylated proteins were resolved by 10% SDS-PAGE, stained with Coomassie blue, dried, and analyzed by autoradiography. Lysates from control and anti-Fas MoAb-treated RPMI-8226 cells were also immunoprecipitated with anti-SAPK Ab (B), anti-p38 MAP kinase Ab (C), or anti-ERK-1 Ab (D). Immune complex kinase assays were performed by addition of GST-Jun (B), GST-ATF-2 (C), or MBP (D) and [γ³²P]ATP and incubation for 15 minutes at 30°C. The phosphorylated proteins were resolved by 10% SDS-PAGE and analyzed by Coomassie blue staining and autoradiography. Anti-SAPK immunocomplexes prepared from control and anti-Fas treated IM-9 (E) and PCL (F ) cells were also assayed for phosphorylation of GST-Jun.