Inducible expression is critical for the function of IκBα but not A20. (A) Simulations of nuclear NF-κB activity in wild-type, iκbα−/−, and a20−/− MEFs in response to TNF. The data are presented as graphs (top) and as heat maps (below) in which the level of NF-κB is color-coded from 0 nM (blue) to 100 nM (red). (B) Simulations of nuclear NF-κB activity in models with altered constitutive transcription rates of IκBα (left) or A20 (right) in the absence of inducible transcription. In each simulation the constitutive transcription rate was multiplied by one of 21 constitutive transcriptional modifiers, ranging from 2−10, 2−9, 2−8. . .1. . .28, 29, 210. The results were graphed over time (X-axis), with the value of the rate modifier on the Y-axis. NF-κB activity is color-coded as in Figure 2A. (C) Simulations of nuclear NF-κB activity in models possessing both constitutive and inducible expression of IκBα (wild type), or each individually. Nuclear NF-κB activity was then measured via EMSA in wild-type cells, or in iκbα−/− cells reconstituted with a constitutively expressing iκbα transgene (pBABE.IκBα.puro) or an empty vector control (pBABE.EV.puro, labeled iκbα−/−) in response to a 15-min pulse of TNF (1 ng/mL). (D) Simulations predict NF-κB activity in TNF-treated cells that have either constitutive or inducible A20 expression, or both (wild type). Nuclear NF-κB activity was measured via EMSA in wild-type cells, or in a20−/− cells reconstituted with a constitutively expressing a20 transgene (pBABE.A20.puro), an NF-κB-inducible transgene (fIL8.A20.puro), or an empty vector control (pBABE.EV.puro, labeled a20−/−) in response to persistent TNF stimulation (1 ng/mL).