Fig. 5: MALT1-dependent proteolytic cleavage inhibits activity and promotes proteasomal degradation of CYLD.

A Immunoblot analysis of CYLD variants in LY10. Cells were transduced with an empty vector (EV) or an expression vector for CYLD (N-terminal fragment, C-terminal fragment, WT or non-cleavable R324A mutant) and sorted for YFP expression. CYLD was detected using an antibody raised against a C‐terminal epitope which detects full-length CYLD and a C‐terminal fragment of CYLD (CYLD‐Ct), or an antibody against an N‐terminal epitope for detection of the N-terminal fragment (CYLD-Nt). β-tubulin was used as loading control. B Flow cytometric analysis of LY10 cells transduced with an empty vector (EV) or a bicistronic expression vector for CYLD (N-terminal fragment, C-terminal fragment, WT or non-cleavable R324A mutant) co-expressing YFP. The percentage of YFP positive cells was followed in time and plotted as the percentage of YFP⁺ cells, normalized to the value at day 3 following retroviral transduction. The mean ± SD of four independent transductions is shown. P > 0.05; ns (non-significant); *P < 0.05; **P < 0.01 using 1-way ANOVA with Tukey’s multiple comparisons test. C Immunoblot analysis of (phosphorylated) IkBα in LY10 transduced with an empty vector (EV) or an expression vector for CYLD (N-terminal fragment, C-terminal fragment, WT or non-cleavable R324A mutant) expressing vector. Three days after sorting cells were incubated with or without 5 µM proteasome inhibitor MG132 for 3 h before harvesting. β-tubulin was used as loading control. D Immunoblot analysis of CYLD cleavage in LY10 and Mino. Cells were incubated with 100 nM ibrutinib for the indicated time points. β-tubulin was used as loading control. E Immunoblot analysis of endogenous CYLD cleavage in LY10 and Mino. Cells were incubated with 100 nM ibrutinib for 24 h in the presence or absence of 10 uM MG132. To prevent apoptosis, cell lines were co-incubated with 10 µM Q-VD-OPh (QVD). β-tubulin was used as loading control.