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Fig. 1. Kinetics of phospho-I{kappa}B{alpha} localization at the T. gondii PVM as an indicator of TgIKK activity. (A-F) IKK{alpha}–/–ß–/– MEFs were cultured on glass coverslips and infected with freshly passaged T. gondii tachyzoites at an m.o.i. of 5:1 for the times indicated. Double immunofluorescence was performed with mouse monoclonal anti-phospho-I{kappa}B{alpha} Ser32 antibody (P-I{kappa}B{alpha}; green) and rabbit polyclonal anti-T. gondii GRA3 antibody (red). Different levels of phospho-I{kappa}B{alpha} coverage at the PVM allowed the classification of vacuoles into three groups: group I showed 0-25% of the PVM covered with phospho-I{kappa}B{alpha} (yellow arrows), group II displayed 50-75% coverage (orange arrows), and group III showed 75-100% of their surface covered with phospho-I{kappa}B{alpha} (blue arrows). Bars, 6 µm. (G-J) Quantification of the different populations of vacuoles indicated a time-dependent increase in phospho-I{kappa}B{alpha} localization at the PVM. WT, IKK{alpha}–/–, IKKß–/– and IKK{alpha}–/–ß–/– MEFs were cultured on glass coverslips and infected with T. gondii at an m.o.i. of 5:1 for the times indicated. A minimum of 300 vacuoles were counted under 100x magnification in a blinded fashion for each cell line and time point. Data represent means ± s.d. of three separate experiments individually quantified by both authors.





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