Figure 9. Ligand-induced conformational change is required for wt but not oncogenic JAK2 to be activated by CK2. (A) Cell lysates from αβ-Ba/F3 cells treated with DMSO (lanes 1–4) or 60μM of TBB (lanes 5–8) prior to GM-CSF deprivation and re-stimulation (0–60 min) were analyzed by immunoblotting using antibodies specific to active-form or total JAK2 proteins. (B) αβ/V617F and HEL cells treated with DMSO (lanes 1and 3) or 60μM of TBB (lanes 2 and 4) were deprived of cytokine for 6 h, and cell lysates were analyzed as described in (A). (C) Wt-JAK2 prepared from αβ-Ba/F3 cells with or without ligand stimulation were treated with increasing doses of proteinase K and then analyzed by immunoblotting using JAK2 antibody. (D) Same as in (C) except that wt or mutant JAK2 prepared from γ2A stable clones were analyzed. (E) Wt JAK2 immunoprecipitated from starved or GM-CSF stimulated αβ- or αβWI-Ba/F3 cells were incubated with or without recombinant CK2, before they were analyzed by immunoblotting as described in (A). (F) Wt or mutant JAK2 immunoprecipitated from γ2A stable clones were analyzed as described in (E). Bottom panels of (C‒F) show the quantification results (mean ± SEM) from three independent experiments. *, p < 0.05; **p < 0.01; ***p < 0.001.