(A) Computational simulations of NF-κB activation by 40–50% inhibition of protein synthesis over 6 hours at different equilibrium free IκBα turnover rates (0.5x, 1x, 2x). The graph shows calculated nuclear NF-κB activity plotted against time.
(B) Immunoblots for IκBα from whole cell extracts of nfκb^−/− (rela^−/− nfkb1^−/− crel^−/−) cells stably expressing wild-type IκBα or the CK2 phospho-acceptor site mutant (IκBα6M) treated with 10 μg/mL CHX for the indicated times.
(C) EMSA for NF-κB activity in nuclear extracts from iκbα^−/− cells reconstituted with either IκBαWT or IκBα6M treated with 40 J/m² UVC.

(A) Model of NF-κB activation by metabolic stress. Metabolic stress inhibits the synthesis of IκB, which is rapidly degraded in the free state (maintained and potentially altered by homeostatic signals), and bound IκB is degraded through low levels of basal IKK activity (maintained and altered by homeostatic and inflammatory signals).
(B) Computational simulations of NF-κB activation at 8 hrs in response to varying degrees of translational inhibition at different equilibrium free IκB turnover rates (in terms of a multiplier to the wild-type parameter on the z-axis).
(C) Computational simulations of NF-κB activation at 8 hrs in response to varying degrees of translational inhibition at different basal IKK activity levels (in terms of a multiplier to the wild-type parameter on the z-axis).

(A) Computational simulations of NF-κB activation via 40–50% inhibition of protein synthesis over 6 hours at different equilibrium levels of basal IKK activity (0.5x, 1x, 2x). The graph shows calculated nuclear NF-κB activity plotted against time.
(B) In vitro kinase assay to measure basal IKK activity of cytoplasmic extracts from untreated ikkβ ^−/− cells reconstituted with IKKβWT, IKKβAA (S177A/S181A), or IKKβKA (K44A). Immunoblot of IKKα from the IP of the IKK complex (bottom panel).
(C) EMSA for NF-κB activity in nuclear extracts from ikkβ ^−/− cells reconstituted with IKKβWT, IKKβAA, or IKKβKA 8 hours after mock or 40 J/m² UVC treatment.
(D) In vitro IKK kinase assay of cytoplasmic extracts from wild-type MEFs stably expressing IKKβEE (S177E/S181E) or wild-type MEFs transduced with empty vector treated with 1 ng/mL TNF for the indicated times. Immunoblot of IKKβ from the IP of the IKK complex (bottom panel).
(E) EMSA for NF-κB activity in nuclear extracts from wild-type MEFs stably expressing IKKβEE or wild-type MEFs transduced with empty vector treated with 40 J/m² UVC for the indicated times.

(A) Contrasting two computational models representing virtual wild type and mutant cells. The “wild-type” model has a high IκB synthesis rate and free IκB degradation rate. In the “IκB^flux” model, free IκBα degradation is decreased 2000-fold, and the synthesis rate is decreased to such a degree that free and bound IκB levels and ratios are the same as in the “wild-type” model.
(B) Computational simulation of a TNF induced NF-κB activation time course in wild-type (blue line) and IκB^flux (green line) models. The graph shows calculated nuclear NF- κ B activity plotted against time.
(C) Computational simulations to determine the dose responsiveness to translational inhibition of wild-type and IκB^flux mutant models. The graph shows the concentration of nuclear NF-κB activity after and 8-hour time course plotted against the degree of translational inhibition.

(A) Computational simulations of NF-κB activation in response to 40–50% translational inhibition (dashed line), 1 ng/mL LPS-mediated IKK activation (dotted line), or combined induction of 50% translational inhibition and 1 ng/mL LPS-mediated IKK activation (solid line).
(B) EMSA for NF-κB activity of nuclear extracts from wild-type MEFs treated with either 40 J/m² UVC alone (lanes 1–4), 1 ng/mL LPS (“LPS^lo”) alone (lanes 9–12) or co-treated with UV and LPS (lanes 5–8) for the indicated times. Signals were quantitated and graphed relative to signal levels in untreated cells (right panel).
(C) Immunoblots for IκBα from whole cell extracts of wild-type MEFs treated with either UVC, LPS, or co-treated with UV and LPS for the indicated times.
(D) RNase Protection Assay showing the mRNA levels of MIP2 in wild-type MEFs treated with either UVC alone, LPS alone, or co-treated with UV and LPS for the indicated times. mRNA levels for L32 are shown as a loading control (bottom panel).
(E) EMSA for NF-κB activity of nuclear extracts from wild-type MEFs. Cells were pretreated with PBS or 1 ng/mL LPS for 4 hours, then either 1 mM DTT (top panel) or 400 nM thapsigargin (Tg, bottom panel) was added for the indicated times.

IKK is not activated by UV:
(A) In vitro kinase assay of cytoplasmic extracts from wild-type MEFs treated with 0, 0.01, 0.1, or 1 ng/mL TNF for 10 minutes.
(B) In vitro kinase assay of cytoplasmic extracts from wild-type MEFs treated with 40 J/m² UVC radiation for the indicated times
(C) In vitro kinase assay of cytoplasmic extracts from wild-type MEFs 8 hours after treatment with the indicated doses of UVC radiation. Immunoblots for IKKα below each kinase assay confirm equal IP-efficiency and loading of the lanes (bottom panels). UV-induced NF-κB activity requires IKK:
(D) EMSA for NF-κB activity in nuclear extracts from wild-type or ikkβ^−/− cells treated with 40 J/m² UVC for indicated times
(E) EMSA for NF-κB activity in nuclear extracts from iκbα ^−/− cells reconstituted with either wild-type IκBα (IκB αWT) or Ser32/36Ala mutant IκBα (IκBαAA) treated with 40 J/m² UVC for the indicated times. An asterisk (*) indicates a non-specific band.
(F) Tracking the degradation of NF-κB-bound IκB following UV irradiation. iκbα ^−/− cells reconstituted with either IκBαWT or IκBαAA were mock-treated, treated with 60 J/m² UVC, or treated with 1 ng/mL TNF. Whole cell extracts prepared 8 hours after UV treatment or after 10 minutes of TNF treatment were subject to immunoprecipitation with anti-p65 antibody. The resulting immunocomplexes were subject to immunoblot analysis for IκBα and p65, representative of multiple experiments.
(G) Tracking degradation of free IκB following UV irradiation. nfκb^−/− cells (rela^−/− nfkb1^−/− crel^−/−) stably expressing IκBαWT or IκBαAA were treated with 40 J/m² UVC or 10 μg/mL CHX and whole cell extracts prepared at the indicated times were subject to immunoblot analysis for IκBα.

(A) Proposed pathways leading to NF-κB activation by UV irradiation. Left: UV may induce GCN2 and PERK activity, which in turn phosphorylate eIF2α to inhibit translation. Center: UV may activate CK2, which may lead to degradation of free and/or bound IκB. Right: UV may also induce IKK activity, which is the primary signal transducer for inflammatory signals.
(B) Flux calculations of IκB turnover in the steady state of resting MEFs, using the computational model of the IκB-NF-κB signaling module. The schematic indicates four degradation pathways delineated in the model and described in the text. The percentages indicate the fraction of the total IκBα degradation flux that occurs within each of these pathways.
(C) Dose response curve of degrees of translational inhibition (in %) to nuclear NF-κB activity (NF-κBn in nM). A translational inhibition curve (Supplemental Figure 2) was used as an input for the computational model and the nuclear NF-κB (NF-κBn) level after 8 hours was calculated and plotted.
(D) Percentage of bulk protein synthesis inhibition in cells treated with 40 J/m² UVC radiation. Wild-type MEFs were either mock- or UV-treated, and radiolabeled for 30 minutes prior to each respective time point with ³⁵S-labeled methionine. Protein extracts were prepared at the indicated times and the amount of ³⁵S incorporation was measured by scintillation count. The amount of ³⁵S incorporation measured for each UV-treated sample was divided by the amount measured for mock-treated to calculate the percent inhibition. Error bars represent standard error of the mean.