Fig. 5. Infection with T. gondii blocks the phosphorylation of p65/RelA and DNA binding. (A,B and C) Whole cell extracts from HFF cells treated with TNF- (left panels), or infected with T. gondii (right panels) were prepared at times indicated. EMSA was performed to determine whether the degradation of IB yields active NF-B dimers (A). (B) Immunoblots with anti-IB antibody (A, bottom panels) were performed to assess total levels in treated cells. (C) HFFs were labeled with ³²Pi orthophosphate as previously described (Zhong et al., 1997) and were treated with TNF- or infected with T. gondii for 15 minutes. Whole cell extracts were either immunoprecipitated overnight with anti-p65 antibody to determine p65/RelA-specific phosphorylation (upper panel) or separated on SDS-PAGE to determine total cellular phosphorylation (lower panel).