FIG. 3.

Virus infection causes NFAT dephosphorylation and an increase in intracellular calcium levels. (A) Infection of Ar-5 T cells by Sendai virus causes dephosphorylation of NFATp. Nuclear extracts were prepared from Ar-5 T cells stimulated with ionomycin (I) for 30 min or with Sendai virus (Vir) for 2 h, in the presence or absence of CsA as indicated in the figure. Lysates were analyzed by sodium dodecyl sulfate–6% polyacrylamide gel electrophoresis followed by Western blot analysis with the anti-NFATp (67.1) antibody. After ionomycin or virus stimulation of the cells, phosphorylated NFATp (P-NFATp) is dephosphorylated (NFATp) and represented as a size shift in the gel. (B) Virus-inducible NFATp/c binds to the −76-NFAT site. An EMSA using nuclear extracts from unstimulated Ar-5 cells (UN) or cells stimulated with ionomycin (I) for 30 min or with Sendai virus (Vir) for 2 h as indicated in the figure was performed. An ionomycin- and virus-inducible complex binds to the oligonucleotide probe spanning positions −85 to −59 containing the −76-NFAT site. Antibodies to NFATp and to NFATc specifically react with the ionomycin- and virus-inducible complex, since they do not react with complexes bound to an Sp1 oligonucleotide probe (47). (C) Infection of Ar-5 T cells by Sendai virus causes an increase in intracellular calcium levels. Cells were exposed to either Sendai virus or ionomycin or a control carrier (allantoic fluid in which the Sendai virus is prepared) at 0 min. Extracellular calcium was later buffered to 0 mM by the addition of 4 mM EGTA (arrow).