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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Br J Haematol. 2011 Apr 15;153(5):589–598. doi: 10.1111/j.1365-2141.2011.08663.x

Fig 1. ETV6-LYN supports cytokine-independent proliferation of Ba/F3 cells.

Fig 1

(A) Schematic representation of ETV6-LYN and its kinase-dead mutant. The kinase-dead mutant of ETV6-LYN (ETV6-LYN KD) was constructed by replacing the C-terminal portion of ETV6-LYN with that of the kinase-dead mutant of LYN (K275A). (B) Detection of ETV6-LYN protein in BaF3/ETV6-LYN cells. The cytoplasmic proteins recovered from Ba/F3 and BaF3/ETV6-LYN cells were separated by SDS-PAGE under reducing (lanes 1 and 2) and non-reducing (lanes 3 and 4) conditions, and then transferred and probed with an anti-Flag antibody. Arrowheads indicate ETV6-LYN oligomers. (C) Detection of tyrosine phosphorylation of ETV6-LYN. ETV6-LYN and ETV6-LYN KD were immunoprecipitated from IL-3-depleted BaF3/ETV6-LYN and BaF3/ETV6-LYN KD cells, respectively, and detected by Western blotting using anti-phosphotyrosine (4G10) and anti-ETV6 antibodies. (D) Detection of kinase activity of ETV6-LYN. In vitro-translated ETV6-LYN and ETV6-LYN KD proteins were subjected to an in vitro kinase assay and detected by Western blotting using anti-phosphotyrosine and anti-ETV6 antibodies. (E) Proliferation of Ba/F3 cells expressing ETV6-LYN or ETV6-LYN KD mutant. Ba/F3 cells were plated at 5×104/well in 24-microtitre plates in triplicate and cultured with or without 2 ng/ml of IL-3. The number of cells was counted at 24, 48, and 72 h of culture. Data are shown as the mean ± SD (n = 3).