Fig. 3: LUBAC is required for TNFR- but not TCR-induced NF-κB activation in Jurkat T cells.

a–c Expression analyses of LUBAC components HOIP, HOIL-1 and SHARPIN in parental and LUBAC KO Jurkat T cell clones by Western blot. d–f NF-κB-EGFP reporter activation in parental and LUBAC KO Jurkat T cell clones following stimulation with TNF, P/I and CD3/CD28 stimulation for 5 h. Quantification of flow cytometric EGFP analyses was done by calculating the median fluorescence intensity (MFI) from 4 (d, f, e for P/I) or 3 (e for TNF and CD3/CD28) biological replicates. g–i NF-κB signaling was analyzed in parental and KO Jurkat T cells after stimulation with TNF (left panel) and P/I (right panel) for the indicated time points. Western blots showing IκBα phosphorylation and degradation are shown. j Reconstitution of HOIP after transduction in HOIP KO Jurkat T cells analyzed by Western blot. k NF-κB-EGFP reporter activation in HOIP WT and mock transduced HOIP KO Jurkat T cells following TNF, P/I and CD3/CD28 stimulation for 5 h was analyzed by flow cytometry. Quantification of flow cytometric EGFP analyses was done by calculating the median fluorescence intensity (MFI) from 4 biological replicates. l Reconstitution of HOIL-1 after transduction in HOIL-1 KO Jurkat T cells analyzed by Western blot. m NF-κB-EGFP reporter activation and quantification was done as in k from 3 biological replicates. n RT-PCR analyses of NFKBIA/IκBa and TNFAIP3/A20 mRNA in HOIP WT and mock transduced HOIP KO (upper panel) and TRAF6 WT and mock transduced TRAF6 KO (lower panel) Jurkat cells following TNF and CD3/CD28 stimulation for 1 and 4 hours from 3 biological replicates. o NF-κB signaling was evaluated in parental and TRAF6 KO, HOIP KO and TRAF6/HOIP DKO Jurkat T cells without and with P/I stimulation using Western blot analysis. All bars show the means ± SEM (d–f, k, m) or ±SD (n) and p-values were calculated by unpaired two-tailed t-test (k, m), one-way ANOVA combined with Dunnett’s multiple comparisons test (d–f), or two-way ANOVA combined with Sidak’s mutiple comparisons test (n). Only significant p-values (<0.05) are shown. kDa kilodalton.