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. 2019 Jun 25;10:1425. doi: 10.3389/fimmu.2019.01425

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Copyright © 2019 Mitchell, Mercado, Adelaja, Ho, Cheng, Ghosh and Hoffmann.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Type II interferon amplifies weak NFκB activating stimuli by enhancing free IκBα degradation. (A) IκBα translational synthesis rates in naïve and IFNγ-conditioned TEPMs as revealed by ³⁵S-Met pulse assay. Average and standard deviation of three biological replicates are shown. (B) Immunoblot for p-IκBα in TEPMs exposed to either LPS or poly(I:C) with or without IFNγ priming. (C) Immunoblot of “free” IκBα compared to an actin control in MEFs deficient in canonical NFκB proteins RelA, cRel and p50 (termed “nfkb^−/−”) exposed to IFNγ. (D) Free IκBα levels in nfkb^−/− MEFs compared to an actin control. Immunoblot of lysates produced from MEFs exposed to 24 h priming with IFNγ or 4 h treatment with ribosomal inhibitor CHX, and followed by addition of proteasome inhibitor MG132. (E) Predictions from the Simplified Model of NFκB (SiMoN) with low (10% at peak) IKK activity, representative of poly(I:C) (blue), and high (40% at peak) IKK activity, representative of LPS. Values were calculated at 0, 0.5, 1, 2, and 4 h and fit with a smoothing spline for consistency with experimental time points. Free IκBα degradation was modulated from the default value (dashed lines) to 10-fold higher (solid lines) based on quantification of immunoblot in 5B. (F) Time course of NFκB induction (quantitated from EMSAs) in naïve or IFNγ-conditioned TEPMs stimulated with poly(I:C) and LPS. (G) Nuclear NFκB activity calculated using SiMoN as a function of bound IκBα degradation (IKK-activity, x-axis) and free IκBα degradation (colored lines). The blue and red arrows indicates the free IκBα degradation-dependent increase in NFκB activity for low and high IKK activities indicative of poly(I:C) and LPS, respectively. (H) Immunoblots of proteasome activator 28 (PA28) levels in TEPMs following exposure to IFNγ. (I) Immunoblots for IκBα and proteasome activator 28 (PA28) in nfkb^−/− MEFs. Both conditions were repeated following PA28 siRNA-mediated knockdown. (J) Immunoblot of IκBα and PA28 levels in nfkb^−/− MEFs transduced with retroviral transgenes. (K) Coomassie-stained SDS-PAGE showing free IκBα and PA28α/β levels following incubation with increasing amounts of purified 20S proteasome (upper panel) contrasted with GST-ubiquitin levels (lower panel), which serves as a negative control. (B–D) show a dataset representative of at least three biological replicates (H–K) show a dataset representative of two biological replicates. Quantitations are relative to basal or t = 0 activity, which is set to 1.