Stimulation with IL-4 for 10 minutes in the presence of activated Raf-1 is not sufficient to activate JNK. ▵Raf-1:ER cells were stimulated for 16 hours at 37°C in RPMI, 10% FCS with or without 100 nmol/L 4HT. The cells were then stimulated for 10 minutes with IL-3 (3) or IL-4 (4) or left untreated as a control (−). (A) ▵Raf-1:ER was IP with antiestrogen receptor antibodies and kinase activity was determined in vitro by using GST-MEK1 as a substrate. (B) JNK was IP with anti-JNK1 antibodies and kinase activity was determined in vitro by using GST-cJun as a substrate. Phosphorylated proteins were visualized after SDS-PAGE and autoradiography. The membrane was subsequently immunoblotted with an anti-JNK1 antibody to determine equivalency of loading.