Fig. 5

Inhibition of translation stabilises translocation of RelA and IRF3. a RelA (NF-κB) translocation and cytoplasmic IκBα levels in response to LPS or CHX + LPS. WT MEFs were stimulated with 1 μg/ml LPS in the absence or presence of 5 μg/ml CHX, fixed and stained at given time-points with antibodies for RelA and IκBα. Representative excerpt from confocal images show cells at 0 (nt), 30, 90 and 240 min after LPS stimulation. Histograms (n ≥ 700, from a representative experiment out of 2) show the full time course of RelA nuclear translocation, defined for Fig. 2. See Supplementary Data 3 for corresponding uncropped immunostaining images. Scale bar: 50 μm. b–d Protein levels in response to poly(I:C) upon PKR inhibition. WT MEFs were stimulated with 1 μg/ml poly(I:C) for 0 (nt), 2 and 4 h in the absence or presence of imidazolo-oxindole PKR inhibitor (1 μM/ml), C16, added at 1 h prior to poly(I:C) transfection. Culture medium for all conditions contained the C16 solvent DMSO (0.5% final concentration), and was FBS-free to prevent interference with C16. b Whole-cell extracts were analysed using antibodies against total PKR, A20 and IκBα, as well as against a phosphorylated (active) form of PKR (p-Thr451). c Nuclear and cytoplasmic fractions were analysed using antibodies against total RelA and IRF3, as well as against their phosphorylated (active) forms, p-RelA (Ser536) and p-IRF3 (Ser396). d Cells were fixed and immunostained for RelA and IRF3. Scatter plots show nuclear translocations of RelA vs. IRF3 (n = 500) based on confocal images analysis. Percentages indicate fractions of active cells; activity was defined by responding (see also Fig. 2b) with both RelA and IRF3 translocation, as illustrated in a mock plot at the top. See Supplementary Data 4 for corresponding uncropped immunostaining images