Inhibition of autonomous activation of JAK-STAT signaling and induction of apoptosis in primary PV cells by the CK2 inhibitor TBB. (A) Primary PV cells were treated with 10 or 50μM of TBB for 4 hours. The protein concentration of cell lysates was measured in duplicate and 100 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. (B) Primary PV cells were treated with 10 or 50μM TBB for 4 hours. Cell lysates were immunoblotted with the indicated antibodies. The densitometric ratios of P-Y-STAT-3 versus STAT-3, P-Y-STAT-5 versus STAT-5, and P-ERK versus ERK were calculated. The values of lanes 2 and 3 were compared with that of lane 1 (control, no inhibition) and the percentage of inhibition was determined. (C) Primary PV cells were treated with different concentrations of TBB (10-50μM) for the indicated times and cell survival was measured with the WST-1 assay. The value of the untreated sample (8 hours) was arbitrarily set as 1. Experiments were performed in triplicate and error bars show ± SD. *P < .05. (D) Primary PV cells were treated with 10 or 50μM TBB for 24 hours. 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.