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Fig. 2. Kinetic analysis of I{kappa}B{alpha} phosphorylation in total cell populations reveals a requirement for host IKK. WT, IKK{alpha}–/–, IKKß–/– and IKK{alpha}–/–ß–/– MEFs were infected at an m.o.i. of 5:1 for the times indicated. Immunoblots were performed with antibodies against phospho-I{kappa}B{alpha} (Ser32) and I{kappa}B{alpha} as described in the Materials and Methods. Elevated levels of phospho-I{kappa}B{alpha} (P-I{kappa}B{alpha}) were apparent by 1 hour p.i. in WT (A) and IKK{alpha}–/– (B) cells. Phosphorylation of I{kappa}B{alpha} was maximal at 9 hours p.i. (A,B), which is concurrent with the increase in TgIKK activity at the PVM observed in Fig. 1. Contrary to this, only a slight elevation in I{kappa}B{alpha} phosphorylation was detected in IKKß–/– cells (C) and a lack of phosphorylation was observed in IKK{alpha}–/–ß–/– cells (D). The levels of I{kappa}B{alpha} in all cell lines examined were comparable between uninfected (0 hour time point) and T. gondii-infected cells.





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