Fig. 2: The NZF domains in HOIL-1L and SHARPIN cooperatively regulate NF-κB activation mainly by binding to linear ubiquitin chains.

A, F Immunoblot analyses of lysates of WT MEFs and HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins. Data are representative of three independent experiments. B, G HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (5 ng/mL) for the indicated times and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. C, H NF-κB activation in HEK293T cells transiently transfected with the indicated combinations of HOIP, HOIL-1L, and SHARPIN was measured using a luciferase assay. Data are shown as the mean ± s.d. of four (C) or three (H) independent experiments. P values were calculated by one-way ANOVA with Tukey’s post hoc test. D HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (5 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 HOIL-1L WT and SHARPIN WT 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 and **P < 0.01. E HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with FLAG-tagged TNF-α (1 μg/mL) for the indicated times. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted as indicated. The experiments were repeated three times, independently, with similar results. Uncropped western blots are available for this figure in the Supplementary Material.