bpV-mediated nuclear translocation of NFAT.

(A) Jurkat cells were either left untreated or stimulated with PMA (20 ng/mL)/Iono (1 μM) (P/I), or bpV[pic] (10 μM) for 1 hour. Nuclear extracts were then prepared as described in “Materials and methods.” Nuclear extracts from bpV-stimulated Jurkat cells (10 μg) were incubated with γ³²P-labeled murine IL-2–derived NFAT-binding site for 20 minutes in the absence (lane 3) or presence of 10-, 100-, or 200-fold excess of unlabeled NFAT oligonucleotide (lanes 4, 5, and 6, respectively) or 100-fold excess of unlabeled NF-κB oligonucleotide (lane 7). Labeled NFAT oligonucleotides were incubated with nuclear extracts from unstimulated Jurkat cells as a negative control (lane 1) or from PMA/Iono-stimulated Jurkat cells as a positive control (lane 2). Samples were then migrated on a native polyacrylamide gel, dried, and exposed on Kodak X-OMAT film. (B) Nuclear extracts from bpV-stimulated Jurkat cells were either left untreated (lane 2) or preincubated with a panel of polyclonal antisera to NFAT1 (lane 3), NFAT2 (lane 4), or NFAT4 (lane 5) for 30 minutes on ice. Samples were then incubated for 20 minutes with the labeled NFAT oligonucleotide and migrated on a native polyacrylamide gel to be subsequently exposed on Kodak X-OMAT film. Extracts from unstimulated Jurkat cells were used as a negative control (lane 1). (C) Nuclear extracts from bpV-treated starved PBMCs (lanes 2-7) or bpV-treated fresh PBMCs (lanes 9-14) were preincubated with antisera such as preimmune normal rabbit serum (NRS) (lanes 3 and 10), NFAT1 (lanes 4 and 11), NFAT2 (lanes 5 and 12), NFAT4 (lanes 6 and 13), and panNFAT (lanes 7 and 14) and then incubated with the NFAT-labeled probe. Lanes 1 and 8 represent nuclear extracts from untreated starved and fresh PBMCs, respectively. Samples were then migrated on a native polyacrylamide gel, dried, and exposed on Kodak X-OMAT film. Free probe, specific complex and supershifted complexes (SS) are indicated on the left side of each panel.