Raf-1 and IL-4 synergize to activate JNK. (A) ▵Raf-1:ER or (B) ▵Raf-1:ER K70W cells were stimulated for 16 hours at 37°C in RPMI, 10% FCS supplemented with IL-3 (3), IL-4 (4), 100 nmol/L 4HT (4HT), or both IL-4 and 100 nmol/L 4HT (4 + HT). JNK-1 was IP and JNK activity was determined in vitro by using GST-cJun as a substrate. Phosphorylated proteins were visualized after SDS-PAGE and autoradiography. The membranes were immunoblotted with anti-JNK antibodies to assess equivalency of loading (JNK).