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. Author manuscript; available in PMC: 2011 Nov 16.
Published in final edited form as: Cancer Cell. 2010 Nov 16;18(5):423–435. doi: 10.1016/j.ccr.2010.10.025

Figure 6. Roles of Jak2 and Src in Mediating rHuEPO-Induced Cell Signaling in HER2 and EpoR Dual-positive Breast Cancer Cells.

Figure 6

(A and B) Dependence of rHuEPO-induced activation of cell signaling on Jak2 activity and expression. MCF7-HER18 cells were pre-exposed to 50 μM AG490 or DMSO in low-serum medium overnight (A) or subjected to expression knockdown of Jak2 by transient transfection with one of two different Jak2 shRNA constructs or control vector for 72 hours (B). The cells were then stimulated with 10 U/ml rHuEPO for 30 minutes, followed by cell lysis and Western blot analysis with the indicated antibodies.

(C) Increased associations between Src and Jak2 and between Src and HER2 after rHuEPO stimulation. MCF7-HER18 and MDA453β cells were treated or untreated with 10 U/ml rHuEPO for 30 minutes or not. Cell lysates were subjected to immunoprecipitation with a Src antibody or mock antibody, followed by Western blot analysis with antibodies direct against Jak2 and HER2. (D and E) Role of Jak2 in rHuEPO-induced associations between Src and EpoR and between Src and HER2, and in rHuEPO-induced Src activation. MCF7-HER18 cells were transiently transfected with Jak2 shRNA constructs or control vector as described in (B) and then stimulated with 10 U/ml rHuEPO for 30 minutes. Src-immunoprecipitates (D) and whole cell lysates (E) were subjected to Western blot analysis with the indicated antibodies. See also Figure S4.