Fig 1: CAP-Gly3 of CYLD is required for regulation of Ub deposition and signaling outcome(A) Relative levels of CXCL8 mRNA in U2OS/NOD2 cell lines treated or not for 3 h with 200 ng/mL L18-MDP. mRNA levels are expressed relative to L18-MDP-treated CYLD KO cells reconstituted with WT CYLD. Data represent mean ± SEM of three to eight independent experiments.(B) Histogram plots showing intracellular flow cytometry analysis of CXCL8-positive U2OS/NOD2 cell lines following stimulation with L18-MDP for 5 h in the presence of brefeldin A and monensin to block secretion of CXCL8.(C) CXCL8 production in U2OS/NOD2 cell lines as in (B). Data represent mean ± SEM of three independent experiments.(D) Purification and western blot analysis of Ub conjugates from U2OS/NOD2 cell lines treated for 1 h with CpA (1 µM) or DMSO prior to stimulation with L18-MDP for 1 h as indicated. **p < 0.01, ***p < 0.001; n.s., not significant.