Inhibition of autonomous activation of JAK-STAT signaling and induction of apoptosis in HEL cells by the CK2 inhibitor TBB. (A) Lysates of HEL cells were immunoprecipitated with anti-JAK2 and R-IgG and then immunoblotted with the indicated antibodies. (B) HEL cells were treated with different concentrations of TBB (10-50μM) for 4 hours. The protein concentration of cell lysates was measured in duplicate, and 65 μg of total protein was analyzed for JAK2 pYpY 1007/1008 expression with ELISA, and then normalized to JAK2 expression. The value of the untreated sample was arbitrarily set as 1. Three independent experiments were performed and error bars show ± SD. *P < .05. (C) HEL cells were treated with different concentrations of TBB (10-50μM) for 4 hours. Cell lysates were immunoblotted with the indicated antibodies. (D-F) HEL cells were treated with different concentrations of TBB (5-25μM) for 24 hours. (D) Cells were stained with annexin V and propidium iodide and examined by flow cytometry. Experiments were performed in triplicate and error bars show ± SD. *P < .05. (E) HEL cells were fixed overnight, stained with propidium iodide, and digested with RNase. The percentage of cells in the sub-G1, G1, S, and G2/M phases was examined by flow cytometry. (F) Cell lysates were immunoblotted with the indicated antibodies.