1. by Yuka Nagata, Eisuke Nishida, and Kazuo Todokoro
Activation of JNK Signaling Pathway by Erythropoietin, Thrombopoietin, and Interleukin-3
by Yuka Nagata, Eisuke Nishida, and Kazuo Todokoro
Blood
Volume 89(8):
April 15, 1997
©1997 by American Society of Hematology

2. IL-3, Epo, and Tpo activate both JNK1 and JNK2 in in vitro kinase assay.
IL-3, Epo, and Tpo activate both JNK1 and JNK2 in in vitro kinase assay. FDC-P2 cells (upper panel), FD-EPO cells (middle panel), and FD-TPO cells (lower panel) were stimulated with IL-3, Epo, and Tpo, respectively, for the indicated time up to 120 minutes. JNK1 (A) and JNK2 (B) activity was measured in the immunoprecipitates with anti-JNK1– and anti-JNK2–specific antibody and [γ-32P]ATP and GST-c-Jun as a substrate. Arrows indicate the phosphorylated GST-c-Jun (molecular weight, 35 kD).
Yuka Nagata et al. Blood 1997;89:
©1997 by American Society of Hematology

3. IL-3, Epo, and Tpo specifically activate both JNK1 and JNK2.
IL-3, Epo, and Tpo specifically activate both JNK1 and JNK2. FDC-P2 cells (left 2 lanes), FD-EPO cells (middle 2 lanes), and FD-TPO cells (right 2 lanes) were stimulated with IL-3, Epo, or Tpo, respectively, for 0 minutes (−) or 15 minutes (+). In-gel JNK assays were performed in the immunoprecipitates with anti-JNK1 (A) or anti-JNK2 (B) antibody. The immunoprecipitates were separated by SDS-polyacrylamide gels containing GST-c-Jun and incubated with [γ-32P]ATP after protein renaturation. The phosphorylated GST-c-Jun corresponding to the molecular weights of JNK1 (46 kD) and JNK2 (55 kD) are indicated by arrows.
Yuka Nagata et al. Blood 1997;89:
©1997 by American Society of Hematology

4. Phosphorylation of SEK1/MKK4 was not induced by IL-3, Epo, or Tpo stimulation.
Phosphorylation of SEK1/MKK4 was not induced by IL-3, Epo, or Tpo stimulation. FDC-P2 cells (A and D), FD-EPO cells (B), and FD-TPO cells (C) were stimulated with IL-3 (A), Epo (B), and Tpo (C), respectively, for the indicated time up to 60 minutes or stimulated with (+) or without (−) sorbitol (D) for 30 minutes. Total cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho–specific SEK1/MKK4 antibody. An arrow indicates the phosphorylated SEK1 (46 kD).
Yuka Nagata et al. Blood 1997;89:
©1997 by American Society of Hematology

5. IL-3, Epo, and Tpo did not activate SEK1/MKK4.
IL-3, Epo, and Tpo did not activate SEK1/MKK4. FDC-P2 cells (lanes 1, 2, 7, and 8), FD-EPO cells (lanes 3 and 4), and FD-TPO cells (lanes 5 and 6) were stimulated with (+) or without (−) IL-3 (lanes 1 and 2), Epo (lanes 3 and 4), Tpo (lanes 5 and 6), and sorbitol (lanes 7 and 8), respectively. SEK1/MKK4 activity was measured in the immunoprecipitates with anti-phospho–specific SEK1/MKK4 antibody and [γ-32P]ATP and GST-JNK1. An arrow indicates the phosphorylated GST-JNK1.
Yuka Nagata et al. Blood 1997;89:
©1997 by American Society of Hematology