The absence of CYLD activity leads to reduced LUBAC retention at the TNF-RSC as well as increased Lys63-Ub and reduced Met1-Ub on receptor components
(A–C) Purification and western blot analysis of the TNFR1 complex in THP1 cells with shRNA knockdown of CYLD or mismatch control (A) and U2OS/NOD2 cell lines (B and C) stimulated with biotin-TNF as indicated.
(D) UbiCRest analysis of TNFR1 complexes purified as in (B). Samples were incubated or not with OTULIN and/or OTUD1 as indicated and analyzed by western blotting.