Differential Desensitization to Type I Interferons Figure 5. USP18 is necessary for differential desensitization. (A) Stat1 phosphorylation induced in HLLR1-1.4 cells stimulated for 30 min with IFN a2 (100 pM), IFN b (100 pM) or IFN c (1 ng/ml) in na¨ıve cells and in cells primed with either IFN b (500 pM) or IFN c (10 ng/ml). Cells were primed for 8 hr and maintained without IFN for 16 hr. (B) Stat3 phosphorylation induced in HLLR1-1.4 stimulated for 30 min with with IFN a2 (100 pM), IFN b (100 pM) or hIL-6 (10 ng/ml) in na¨ıve cells and in cells primed with IFN b (500 pM) or hIL-6 (100 ng/ml). Cells were primed for 8 hr and maintained without IFN for 16 hr. Lysates (30 mg) were immunoblotted with the indicated Abs. (C) Level of USP18 mRNA in HLLR1-1.4 cells stimulated for 6 hr with IFN a2, IFN b (500 pM), IFN l1 (50 pM), IFN c (1 ng/ml) or hIL-6 (100 ng/ml) as determined by qRT-PCR. Each sample was run in triplicate. Transcripts were normalized to the level of 18S transcripts. The ratios between treated and untreated samples in each subset are shown, taking as 1 the ratio in untreated samples. (D) Kinetic profile of USP18 induction in HLLR1-1.4 cells stimulated with 100 pM of IFN b or IFN l1 for the indicated times. Cell lysates (30 mg) were immunoblotted with the indicated Abs. The asterisk points to a nonspecific band. (E) USP18 is necessary for differential desensitization. HLLR1-1.4 cells were transfected with a control pool of siRNA (Control siRNA) or a pool of four USP18 targeting siRNA (USP18 siRNA). Twenty four hr after transfection, cells were either left untreated (na¨ıve) or primed for 8 hr with the indicated IFN. After 16 hr of resting, cells were stimulated for 30 min with 100 pM of IFN a2 or IFN b. Cell lysates (30 mg) were analysed with the indicated antibodies. The asterisk in the bottom panel points to a band cross-reacting with anti-USP18 Abs (see also USP18 blot in Fig. 3C). Individual USP18 targeting siRNA were also used with similar results (data not shown). doi:10.1371/journal.pone.0022200.g005 PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22200