CK2 is required for OSM-induced JAK2 and JAK1 activation. (A) MEFs were transfected with nontarget (NT) siRNA (100nM), CK2α siRNA (100nM), CK2β siRNA (100nM), or CK2α (50nM) plus CK2β siRNA (50nM) for 48 hours and then stimulated with 5 ng/mL of OSM for 10 minutes. (B-C) MEFs were pretreated with TBB (50μM) or emodin (50μM) for 2 hours and then stimulated with 5 ng/mL OSM for 10 minutes. (A-B) The protein concentration of the cell lysates was measured in duplicate and 65 μg of total protein was analyzed for JAK2 pYpY 1007/1008 expression by ELISA and normalized to JAK2 expression, which is not affected by CK2 siRNAs. The value of the untreated sample was arbitrarily set as 1. Three independent experiments were performed and error bars show ± SD. *P < .05. Cell lysates were immunoblotted with the indicated antibodies (lower panels). (C) Cell lysates were blotted with the indicated antibodies. (D) MEFs were pretreated with TBB (50μM) for 2 hours and then stimulated with 10 ng/mL of OSM for 30 minutes. Lysates were immunoprecipitated with anti-gp130 antibody and analyzed by immunoblotting. The blot was detected with anti-phosphotyrosine antibody and then reprobed with gp130 antibody after stripping. (E) MEFs were pretreated with TBB (50μM) or emodin (50μM) for 2 hours and then stimulated with 0.1 ng/mL of OSM for 30 minutes. Cell lysates were immunoblotted with the indicated antibodies.