Detection of NF-P motif–binding factors in the cultured spleen cells.

Arrowheads 1 and 2 indicate the same positions of protein-DNA complexes as shown in Figure 6. (A) Two nuclear factors that specifically bind with the NF-P motif are present in the nucleus oftg/tg–cultured spleen cells. The nuclear extract (NE) was prepared from tg/tg spleen cells after culturing with rmIL-2 for 10 days and incubated with ³²P-labeled oligonucleotide NF in the presence or absence (−) of the competitor. For competition, NE was incubated with an excess amount (100-fold) of unlabeled oligonucleotide NF or oligonucleotide mNF prior to the reaction with the probe. The reaction mixture was separated by polyacrylamide gel. (B) Detection of the NF-P motif–binding factors in the nuclear and cytoplasmic (CE) extracts by EGMSA. Each cell extract was prepared from CTLL-2 cells, and +/+, mi/mi, and tg/tg spleen cells after culturing with rmIL-2 for 10 days. The extracts were incubated with ³²P-labeled oligonucleotide NF and separated on polyacrylamide gel (upper panel). Aliquots (10 μg per lane) of the extracts were blotted with the PCNA and pan-actin antibodies (lower panels). (C) Detection of the NF-P motif–binding factors during in vitro development of mi/mi NK cells. To produce NK cells, spleen cells from mi/mi mice were cultured as described in “Materials and methods.” The nuclear and cytoplasmic fractions were extracted from the cultured cells on the indicated days after the initiation of the culture. The extracts were incubated with³²P-labeled oligonucleotide NF and separated on polyacrylamide gel.