Fig. 1: The ubiquitin-binding activity of HOIL-1L NZF preferentially contributes to NF-κB activation.

A Schematic illustration of the domains of LUBAC subunits. B Immunoblot analyses of lysates of WT MEFs and HOIL-1L-null MEFs stably reconstituted with the indicated proteins. Data are representative of three independent experiments. C, E WT MEFs and HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL) (C) or TNF-α (2.5 ng/mL) plus CHX (20 μg/mL) (E) for the indicated times, and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. D HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL), and expression of NF-κB target genes was measured by quantitative PCR. All gene expression levels are normalized against the corresponding levels of ACTB and expressed as fold changes relative to cells with an empty vector at 0 h. Data are shown as the mean ± s.d. of three independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. F HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) and CHX (20 μg/mL) for the indicated times after pretreatment with Z-VAD-FMK (10 μM) for 1 h, and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. G, H HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) and CHX (20 μg/mL), and cell viability was continuously measured using the xCELLigence system. A representative image (G) and statistical analyses of the normalized cell index at 4 h (H). Data are shown as the mean ± s.d. of three independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. Uncropped western blots are available for this figure in the Supplementary Material.