Dose-dependent activation of JNK in ▵Raf-1:ER but not Ba/F3 cells after stimulation with 4HT for 16 hours. (A) ▵Raf-1:ER cells were stimulated at 37°C for 16 hours at 2 × 10⁵cells/mL in RPMI, 10% FCS plus IL-3, IL-4, or IL-4 plus increasing concentrations of 4HT. (B) Ba/F3 cells were stimulated for 16 hours at 37°C in RPMI supplemented with 10% FCS and IL-3 (3), IL-4 (4), or IL-4 plus 100 nmol/L 4HT (4 + HT). JNK was IP from the cell lysate, and kinase activity was determined in vitro by using GST-cJun as a substrate. Phosphorylated proteins were visualized after SDS-PAGE and autoradiography. The quantity of immunoprecipitated protein in each lane was assessed by immunoblotting (IB) with antibodies against JNK (JNK).