Fig. 2. Kinetic analysis of IB phosphorylation in total cell populations reveals a requirement for host IKK. WT, IKK^–/–, IKKß^–/– and IKK^–/–ß^–/– MEFs were infected at an m.o.i. of 5:1 for the times indicated. Immunoblots were performed with antibodies against phospho-IB (Ser32) and IB as described in the Materials and Methods. Elevated levels of phospho-IB (P-IB) were apparent by 1 hour p.i. in WT (A) and IKK^–/– (B) cells. Phosphorylation of IB 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 IB phosphorylation was detected in IKKß^–/– cells (C) and a lack of phosphorylation was observed in IKK^–/–ß^–/– cells (D). The levels of IB in all cell lines examined were comparable between uninfected (0 hour time point) and T. gondii-infected cells.