Monokine-dependent Hlx expression by human CD56^bright NK cells is delayed with respect to IFN-γ transcription. (A) Delayed induction of Hlx protein with respect to IFN-γ production in primary NK cells. (upper) Total NK cells were stimulated with IL-12/IL-18 for indicated time points, and Hlx protein levels were analyzed by immunoblotting. β-Actin levels were analyzed to ensure equal loading (n = 4 experiments). (lower) In parallel, IFN-γ mRNA levels were analyzed by QRT-PCR. Mean ± SEM from 4 separate experiments is shown. (B) Maximal Hlx protein induction requires monokine costimulation. Total NK cells were stimulated for 72 hours, as indicated, and Hlx and β-actin protein levels were analyzed by immunoblotting (n = 3 experiments). (C) Preferential induction of Hlx protein in the CD56^bright NK subset. (upper) FACS-purified unstimulated NK subsets were lysed directly (UN) or after stimulation with the indicated monokine combinations (72 hours). Lysates were analyzed for Hlx and β-actin protein by immunoblotting (n = 3 experiments). (lower) FACS-purified NK subsets were lysed immediately for RNA (UN) or stimulated with IL-12/IL-15 for 12 hours before lysis. Hlx mRNA levels were analyzed by QRT-PCR. Mean ± SEM from 5 separate donors is shown.