Fig. 2.

Quantification of NF-κB, IκBα and A20 levels in response to TNFα and LPS stimulation. a Immunostaining time profiles of RelA (NF-κB) nuclear/total ratio and total IκBα/total RelA (NF-κB) ratio (in arbitrary units) in response to 1 μg/ml LPS with 5 μg/ml CHX costimulation started 1 h before LPS. Open squares show values calculated in each of five confocal frames analysed. Filled squares show mean over these five frames containing in total more than 500 cells for each time point. b Histograms showing nuclear RelA (NF-κB) fluorescence normalized to cell average fluorescence for unstimulated cells (grey) and 1 h after 1 μg/ml LPS with 5 μg/ml CHX costimulation (green); see Methods for details of normalization. Coefficient μ is the histogram average while σ is the standard deviation. In stochastic numerical simulations, total single-cell NF-κB levels were drawn at random based on the data used to plot the histogram; see Methods. Bottom subpanel shows cumulative distributions for unstimulated (grey line) and stimulated (green line) cells. Kolmogorov–Smirnov statistic (K–S) equals 0.878, which implies that at least 87.8 % of cells respond to stimulation. c Experimental IκBα and A20 mRNA time profiles after 10 ng/ml TNFα and 1 μg/ml LPS stimulation from three independent measurements. Data show absolute quantification by digital PCR for TNFα stimulation or rescaled RT-PCR quantification using digital PCR measurements in selected time points. Model simulated mRNA profiles after 10 ng/ml TNFα show the average over 300 stochastic simulations. The numerical values are shown only for experimental time points, and are connected by line only to guide the eye. d Western blot analysis of cytoplasmic and nuclear fractions of RelA (NF-κB), IκBα and A20. Blots from one of three quantified experiments are shown. Nuclear IκBα and A20 were near the limit of detection. Model simulated protein profiles after 10 ng/ml TNFα show the average over 300 stochastic simulations