Fig 2. ETV6-LYN activates STAT5 in a JAK2-independent manner.

(A) Signalling pathways activated by ETV6-LYN in Ba/F3 cells. Parental Ba/F3 were deprived of IL-3 for 12 h and then further incubated with or without IL-3 for 15 min. BaF3/ETV6-LYN cells were deprived of IL-3 for 12 h. The cells were lysed and the phosphorylation status of JAK2, STAT5, STAT3, Akt, Erk1/2, p38, and JNK were examined by Western blotting. To detect Erk1/2, p38, and Akt, equal amount of whole cell lysate were used and probed with anti-phospho-Erk1/2, anti-phospho-p38, or anti-phospho-Akt antibody (upper panels), and reprobed with anti-Erk1/2, anti-p38, or anti-Akt antibody (lower panels). To detect JNK, JAK2, STAT3 and STAT5 activation, JNK, JAK2, STAT3 or STAT5 proteins were immunoprecipitated and probed with anti-phospho-JNK, anti-phospho-JAK2, or anti-phosphotyrosine antibody, and reprobed with anti-JNK, anti-JAK2, anti-STAT3, or anti-STAT5 antibody. (B) Kinase activity of ETV6-LYN KD in Ba/F3 cells. Parental Ba/F3 cells were deprived of IL-3 for 12 h and then further incubated with or without IL-3 for 15 min. BaF3/ETV6-LYN and BaF3/ETV6-LYN KD cells were deprived of IL-3 for 12 h. The cells were lysed and the phosphorylation status of STAT5 and Akt was examined by Western blotting as in (A). (C) STAT5 activation by ETV6-LYN vs. canonical cytokine signalling pathway. BaF3/ETV6-LYN cells were treated with dasatinib or imatinib with or without IL-3. The phosphorylation status of STAT5 was examined as in (A).