Failure to detect an autocrine factor that can activate JNK in a cell-mixing experiment. Cells (1 × 10⁶ Ba/F3) expressing GST-JNK1 were mixed together with 1 × 10⁷▵Raf-1:ER cells and stimulated for 16 hours at 37°C in RMPI, 10% FCS with either IL-3 (3), IL-4 (4), or IL-4 plus 100 nmol/L 4HT (4 + HT). (A) GST-JNK1 was affinity purified (AP) from the mixed cell lysate with glutathione Sepharose beads, and the activity was determined in vitro assay by using GST-cJun as a substrate. After SDS-PAGE and autoradiography, the membrane was immunoblotted with antibodies against GST to assess equivalency of loading (GST). (B) After the lysate had been cleared with glutathione Sepharose, endogenous JNK was IP with anti-JNK1 antibodies, and the kinase activity was determined in vitro by using GST-cJun as a substrate. After SDS-PAGE and autoradiography, the membrane was immunoblotted with antibodies against JNK1 (JNK) to assess equivalency of loading.