Fig. 7. Localization of P-IB to the T. gondii PVM. MEFs grown on glass coverslips were infected at a m.o.i. of 5:1 for 20 hours. Double immunofluorescence was performed with mouse monoclonal anti-P-IB (green) and mouse monoclonal anti-T.-gondii SAG1 (red). A distinctive pattern of P-IB was observed around the T. gondii PVM (A,C, white arrows). Uninfected cells (D, black arrows) displayed little, if any, cytoplasmic P-IB staining. Blocking experiments with a peptide corresponding to a short amino acid sequence containing phosphorylated Ser 32 of IB eliminated labeling by the anti-P-IB antibody but had no effect on the parasite marker SAG1 (E-G). Treatment with TNF resulted in high levels of P-IB in both infected and uninfected cells (I,K). Staining for P-IB was observed in the host cell cytoplasm, nucleus and around the PVM. Incubation with the blocking peptide resulted in the loss of P-IB staining both at the PVM and host cell (M-O). Scale bar, 20 µm.