Fig. 3. T. gondii infection results in an increase in NF-B DNA binding activity. (A) Binding reactions for EMSAs were performed with a radiolabeled oligonucleotide probe containing the NF-B consensus sequence and nuclear protein extracts from uninfected, infected or TNF-treated wild-type (WT) MEFs. Antibodies to NF-B members p50, p65, RelB, c-Rel and p52 were used for supershift assays. An increase in NF-B binding activity was observed in infected MEFs and TNF-treated cells compared with uninfected controls. Supershifts of NF-B complexes confirmed the presence of p50/p65 heterodimers in infected and TNF-treated cells. Supershifts were also observed with antibodies to RelB and p52 in infected cells. (B) Analysis of NF-B translocation by EMSA in p65^–/– MEFs infected with T. gondii. Nuclear extracts from uninfected, infected and TNF-treated p65^–/– cells revealed similar levels of NF-B binding activity. Supershift assays of NF-B complexes showed the presence of p50/p50 homodimers in the three experimental conditions, but p52 was only observed in T.-gondii-infected cells. As expected, no supershift was detected with anti-p65 antibodies. Abbreviation: n.s., non-specific complex.

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