FIGURE 4. IKK activity.

IKK activity was assessed using recombinant GST-IκBα-(1–54) and [γ-³²P]ATP as substrates. The phosphorylated GST fusion protein was detected by autoradiography. a, IKK activity was determined in NIH-OVCAR-3 cells co-transfected with IHPK2 (wild type or mutants) and TRAF2, followed by TNF-α stimulation for 15 min. Cells expressing the double mutant did not show significant differences in IKK activity compared with cells expressing wild type IHPK2. Untreated cells typically exhibit 5–10% the activity of stimulated cells (31). Expression of IHPK2 transgene is indicated, and GAPDH served as loading control. The experiment was performed three times with similar results. b, Coomassie blue-stained gel demonstrated equal loading of GST-IκBα-(1–54) substrate utilized in the kinase assay.