Fig. 1: Comparison of TRAF6 and LUBAC-dependent CBM signaling in human CD4+ T cells.

a TRAF6 and HOIP expression normalized to β-Actin in CD4+ T cells from different donors after sgRNA transfection quantified based on Western blotting from 7 biological replicates (see Supplementary Figs. 1c, 2a). b Western blot analysis of sgControl, sgTRAF6, sgHOIP and sgHOIL-1 KO primary human CD4+ T cells after stimulation with P/I for the indicated time points. Expression of the target genes (TRAF6, HOIP and HOIL-1), NF-κB signaling and MALT1 substrate cleavage were evaluated by Western blot. c, d Representative flow cytometric analyses (left panel) and quantification of changes in mean fluorescence intensity (MFI; values normalized to untreated [UT], right panel) of IκBα levels in sgControl, sgTRAF6, sgHOIP and sgHOIL-1 primary CD4+ T cells after P/I (c) or TNF (d) stimulation (30 min) from 8 (sgControl) and 4 (targeting sgRNA) biological replicates. IκBα levels of UT samples were set to 1. Treated samples were normalized to the untreated samples. e Representative flow cytometric analyses (left panel) and quantification of changes in MFI (right panel) of p-p65 levels in sgControl, sgTRAF6, sgHOIP and sgHOIL-1 primary CD4 T cells after P/I stimulation (30 min) shown for donor A from 8 (sgControl) and 4 (targeting sgRNA) biological replicates. f Image stream analyses showing representative pictures. Scale bar: 7 µm. g Histograms showing similarity scores of p65 and NucBlue (nucleus) stain and quantification of p65 and NucBlue similarity scores and percentage nuclear p65 of sgControl, sgTRAF6 and sgHOIP transfected CD4+ T cells after P/I stimulation (30 min) from 3 biological replicates. All bars show the means ± SD and p-values were calculated by unpaired two-tailed t-test (a) one-way ANOVA (c, d, g) or two-way ANOVA (e) combined with Dunnett’s (c–e) or Tukey’s (g) multiple comparisons test. kDa: kilodalton.