Fig. 6: MALT1 cleavage generates a partially active HOIL-1 N-terminal fragment.
From: LUBAC modulates CBM complex functions downstream of TRAF6 in T cells
a Scheme of HOIL-1 WT and mutant proteins. LTM: LUBAC-tethering motif, UBL: Ubiquitin-like, NZF: Npl4-type zinc-finger, RING: Really interesting new gene, IBR: In-between-RING. b LUBAC subunit expression after transduction of HOIL-1 WT and mutants into HOIL-1 KO Jurkat T cells analyzed by Western blot. c NF-κB-EGFP reporter induction in HOIL-1 KO Jurkat T cells with HOIL-1 WT and mutant constructs after P/I, CD3/CD28 and TNF stimulation for 5 h. EGFP expression was analyzed by flow cytometry and quantification of median fluorescence intensity (MFI). d Cell viability determined by CellTiter-Glo assay after 14 h TNF treatment in parental, HOIP KO and HOIL-1 KO Jurkat T cells. e CASP3/7 activation was assessed after 3.5 h TNF treatment in parental, HOIP KO and HOIL-1 KO Jurkat T cells. f Parental and HOIL-1 KO Jurkat T cells were treated as indicated. CASP and PARP cleavage was assessed by Western blot. g Cell viability determined by CellTiter-Glo assay in HOIL-1 KO Jurkat T cells expressing HOIL-1 WT and mutants following 14 h TNF stimulation. h Induction of CASP-8 cleavage in HOIL-1 KO Jurkat T cells after reconstitution with mock or HOIL-1 WT and mutants after 6 h TNF or TNF/CHX stimulation was analyzed by Western blot. i Expression of HOIL-1 WT as well as HOIL-1 N-term and HOIL-1 C-term alone or in combination after transduction into parental (left panel) and HOIL−1 KO (right panel) Jurkat T cells analyzed by Western blot. j Cell viability determined by CellTiter-Glo assay in parental (upper panel) or HOIL-1 KO (lower panel) Jurkat T cells transduced with HOIL-1 WT and mutants after TNF or TNF/CHX stimulation for 14 h. All quantifications were done from 3 (c, d, g, j) or 4 (e) biological replicates after normalization of median fluorescence intensity (MFI; c) or relative luminescence units (RLU; d, e, g, j) to untreated (UT) samples. All bar graphs represent means ± SEM and p-values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test. Only significant p-values (<0.05) are shown. kDa kilodalton.
