, Dose-dependent sensitization of IFNα signal transduction IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.Figure 1IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.5 cells. Cells were seeded 24 hours prior to the start of the experiment. Three hours before stimulation, cells were growth factor-depleted and were subsequently stimulated with the indicated concentrations of IFNα. Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=1 to N=38) using a combined scaling and error model. Data is approximated with a sigmoidal function and signals corresponding to a low dose (2.8 pM IFNα), a medium dose (28 pM IFNα) and a high dose (1400 pM IFNα) are displayed with dashed lines. Experimental design of IFNα memory experiment in Huh7.5. Cells were seeded 24 hours prior to the start of the experiment. Three hours before prestimulation, cells were growth factor-depleted and were subsequently prestimulated with 2.8 pM IFNα, 1400 pM IFNα or were left untreated. After 24 hours, cells were stimulated with 1400 pM IFNα. Cytoplasmic and nuclear protein lysates were collected at indicated time points. Representative immunoblot of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 cells prestimulated for 24 hours with 2.8 pM IFNα, 1400 pM IFNα or without prestimulation. Time points after presimulation are indicated. 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets. Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. An asterisk indicates pSTAT1β. Calnexin served as loading control. Molecular weights are indicated on the left. Immunoblot detection was performed with chemiluminescence employing a CCD camera device (ImageQuant).   Quantification of immunoblots of IFNα-induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7.5 cells prestimulated with 2.8 pM IFNα, 1400 pM IFNα or without pretreatment. Time points after presimulation are displayed. Data is displayed as fold change relative to untreated cells. Errors were estimated with a combined scaling and error model, comprising 1σ confidence interval estimated from biological replicates (N=3). Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 1400 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points displayed as dots with 1σ confidence-interval estimated from biological replicates (N=4 to N=14) using a combined scaling and error model. Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 2.8 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=4 to N=6) using a combined scaling and error model. Dashed lines indicate smoothing splines.