Fig. 1. T. gondii infection induces nuclear translocation of NF-B. MEFs were incubated in medium alone (uninfected), treated with 20 ng ml^–1 of TNF for 20 minutes or infected with T. gondii at an m.o.i. of 5:1 for 20 hours. Confocal immunofluorescence microscopy was performed with primary rabbit polyclonal antibodies against NF-B p50 (A) and p65 (B), and mouse monoclonal anti-T.-gondii SAG1. Proteins were localized using species-specific Oregon Green (NF-B) or Texas Red (SAG1) conjugated secondary antibodies. Uninfected cells displayed mainly cytoplasmic staining of p50 and p65 (A,B, top, respectively). Stimulation with TNF caused translocation of both subunits to the nucleus. Infection with T. gondii resulted in nuclear localization of p50 and p65 (A,B, middle, respectively). Uninfected cells in the same field failed to exhibit NF-B translocation. Treatment of the mixed population of uninfected and infected MEFs resulted in translocation of p50 and p65 in all cells (A,B, bottom, respectively). Scale bar, 20 µm. (C) NF-B translocation induced by T. gondii was confirmed in nuclear fractions of infected MEFs by immunoblotting. Cells were infected at increasing m.o.i. for 20 hours. Treatment of uninfected cells with TNF (20 ng ml^–1, 20 minutes) was used as a positive control. Nuclear fractions were prepared as described in the Methods section and immunoblots were performed with polyclonal rabbit antibodies to p50 and p65. A dose-dependent increase in host cell nuclear p50 and p65 localization was observed in response to T. gondii infection.