Epo rapidly and transiently activates JNK1 and JNK2 during Epo-induced erythroid differentiation. SKT6 cells were stimulated with Epo for 0, 3, 5, 15, 30, 60, and 180 minutes (lanes 1 through 7) and for 1, 2, 3, and 4.5 days (lanes 8 through 11). Activity of JNK1 (A) and JNK2 (B) was assayed in the immunoprecipitates with anti-JNK1– or anti-JNK2–specific antibody and GST-c-Jun as a substrate. Arrows indicate the phosphorylated GST-c-Jun (molecular weight, 35 kD). Lower panels show the immunoblots of the immunoprecipitated JNK1 or JNK2 in each lane. (C) In-gel kinase assay of JNK1 and JNK2. Cell lysates before (−) and 5 minutes after (+) Epo stimulation were used for in-gel kinase assay. Arrows indicate the position of JNK1 (molecular weight, 46 kD) and JNK2 (molecular weight, 55 kD).