Figure 1
Figure 1. CK2 is required for OSM-induced STAT activation and gene expression. (A-E) Cell lysates were immunoblotted with the indicated antibodies. (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, then stimulated with 1 ng/mL of OSM for 10 minutes. The densitometric ratios of p-Y-STAT-3 versus STAT-3 were calculated. The values shown in lanes 4, 6, and 8 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (B) MEFs were pretreated with 25-100μM TBB for 2 hours and then stimulated with 1 ng/mL of OSM for 10 minutes. The densitometric ratios of p-Y-STAT-3 versus STAT-3 were calculated. The values shown in lanes 3, 4, and 5 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (C) MEFs were pretreated with TBB (50μM) for 2 and 4 hours and then stimulated with 0.1 ng/mL of OSM for 30 minutes. (D) MEFs were transfected with HA-CK2α or CK2α-inhibitor–resistant constructs for 24 hours, pretreated with TBB (50μM) for 2 hours and then stimulated with 1 ng/mL of OSM for 30 minutes. (E) MEFs were pretreated with CK2 inhibitors for 2 hours and then treated with OSM (10 ng/mL) for 30 minutes. (F) MEFs were transfected with nontarget (NT) siRNA (100nM), CK2α siRNA (100nM), CK2β siRNA (100nM), or CK2α (50nM) plus CK2β siRNA (50nM) for 48 hours, then stimulated with 0.1 ng/mL of OSM for 30 minutes. RNA was prepared and analyzed by RT-PCR for expression of SOCS-3 and GAPDH. The densitometric ratios of SOCS-3 versus GAPDH were calculated. The values of lanes 4, 6, and 8 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (G) MEFs were pretreated with TBB (50μM) for 2 hours and then stimulated with different concentrations of OSM (0.1-0.5 ng/mL). Total mRNA was extracted and analyzed by RPA with probes specific to SOCS-3 and GAPDH (loading control). The densitometric ratios of SOCS-3 versus GAPDH were calculated. The value of lane 4 was compared with that of lane 3 (control, no inhibition), the value of lane 6 was compared with that of lane 5, and the percentage of inhibition was determined.

CK2 is required for OSM-induced STAT activation and gene expression. (A-E) Cell lysates were immunoblotted with the indicated antibodies. (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, then stimulated with 1 ng/mL of OSM for 10 minutes. The densitometric ratios of p-Y-STAT-3 versus STAT-3 were calculated. The values shown in lanes 4, 6, and 8 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (B) MEFs were pretreated with 25-100μM TBB for 2 hours and then stimulated with 1 ng/mL of OSM for 10 minutes. The densitometric ratios of p-Y-STAT-3 versus STAT-3 were calculated. The values shown in lanes 3, 4, and 5 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (C) MEFs were pretreated with TBB (50μM) for 2 and 4 hours and then stimulated with 0.1 ng/mL of OSM for 30 minutes. (D) MEFs were transfected with HA-CK2α or CK2α-inhibitor–resistant constructs for 24 hours, pretreated with TBB (50μM) for 2 hours and then stimulated with 1 ng/mL of OSM for 30 minutes. (E) MEFs were pretreated with CK2 inhibitors for 2 hours and then treated with OSM (10 ng/mL) for 30 minutes. (F) MEFs were transfected with nontarget (NT) siRNA (100nM), CK2α siRNA (100nM), CK2β siRNA (100nM), or CK2α (50nM) plus CK2β siRNA (50nM) for 48 hours, then stimulated with 0.1 ng/mL of OSM for 30 minutes. RNA was prepared and analyzed by RT-PCR for expression of SOCS-3 and GAPDH. The densitometric ratios of SOCS-3 versus GAPDH were calculated. The values of lanes 4, 6, and 8 were compared with that of lane 2 (control, no inhibition) and the percentage of inhibition was determined. (G) MEFs were pretreated with TBB (50μM) for 2 hours and then stimulated with different concentrations of OSM (0.1-0.5 ng/mL). Total mRNA was extracted and analyzed by RPA with probes specific to SOCS-3 and GAPDH (loading control). The densitometric ratios of SOCS-3 versus GAPDH were calculated. The value of lane 4 was compared with that of lane 3 (control, no inhibition), the value of lane 6 was compared with that of lane 5, and the percentage of inhibition was determined.

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