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. 2020 Jul 21;16(7):e8955. doi: 10.15252/msb.20198955

Figure 3. Model calibration with IFNα‐induced signal transduction in Huh7.5 upon prestimulation and stimulation with IFNα.

Figure 3

Growth factor‐depleted Huh7.5 cells were prestimulated with 2.8, 28, 1,400 pM IFNα or left untreated and were stimulated with 1,400 pM IFNα 24 h later. IFNα‐induced signaling was measured by time‐resolved quantitative immunoblotting and detected with chemiluminescence using a CCD camera‐based device. Data were normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.
  1. Model calibration with time‐resolved IFNα‐induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data were represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N = 3 to N = 23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.
  2. Model calibration with time‐resolved IFNα‐induced feedback transcripts upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα, assessed by qRT‐PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT, and TBP. Experimental data are represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N = 3 to N = 14) using a combined scaling and error model. Model trajectories are represented by lines.
  3. Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9, and USP18 determined 24 h after prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Calibrator proteins were spiked into 10 μg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD camera‐based device. Averaged values (N = 4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.
  4. Model calibration of IFNα‐induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8, 28, or 1,400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data are represented by filled circles with errors representing 1σ confidence interval estimated from biological replicates (N = 4 to N = 22) using a combined scaling and error model. Model trajectories are represented by lines.