Evaluation of mRNA for IFN-γ. (A) IFN-γ message was analyzed. Liver-enriched NK cells (greater than 90% NK1.1⁺) were left untreated (▵) or coated with anti-Ly49D at 4°C for 15 minutes, washed, and pretreated at 37°C with IL-12. Cells were washed, actinomycin D was added, and cell lysates were evaluated for IFN-γ mRNA. ▴ indicates anti-Ly49D + actinomycin D; ○, IL-12 + actinomycin D; and •, anti-Ly49D + IL-12 + actinomycin D. Values are expressed relative to mRNA level at 30 minutes before actinomycin D addition (100%), and were derived from digital evaluation of RPA for IFN-γ, relative to L32 control RNA. Values are representative of 2 experiments. (B) Nuclear and cytoplasmic mRNA for IFNγ were analyzed. NK cells were lysed and fractionated, as described in “Materials and methods.” RPA was performed on nuclear and cytoplasmic RNAs using [³³P]UTP-labeled exon 1/intron 1 (E1-I1), exon 3/intron 3 (E3-I3), and L32 rRNA riboprobes. The exon/intron probes hybridized and protected unspliced nuclear IFN-γ pre-mRNA, whereas the exon portion of the exon/intron probes recognized only the spliced form of the IFN-γ mRNA in the nucleus and the cytoplasm. L32 was used as a control for RNA input between samples. (Results from a representative RPA are shown in Figure S2). Lanes from left to right represent the following: nontreated (NT), LY49D cross-linked (49D), IL-12-activated (IL-12), and LY49D plus IL-12 (D/12) coactivated NK cells. Graph representation of the quantitation performed by ImageQuaNT analysis on the image shown in panel B. Normalized mRNA values corresponded to the accumulation of exon/intron and exon-only IFN-γ mRNA relative to L32 mRNA in the nuclear (top) and cytoplasmic (bottom) compartments.