Figure 1. MIB2 interacts with CYLD.

(A) MIB2 interacts with CYLD in GST pull down assays. The ubiquitin hydrolase catalytic domain of CYLD (CYLD^USP) was expressed as a fusion protein with glutathione-S-transferase (GST) in HEK-293T cells and CYLD-associated proteins were isolated from whole cell lysates using a GST pull down assay. MIB2 co-purified with recombinant GST-CYLD ubiquitin specific protease domain (GST-CYLD^USP) and was identified by subjecting excised bands to tryptic digest followed by matrix assisted laser desorption and tandem mass spectrophotometry (also see Supplementary Table 1). (B) Endogenous immunoprecipitation of MIB2 by CYLD. Control rat IgG or rat anti-CYLD monoclonal antibody (2 μg) were incubated with HEK-293T lysates. After immunoprecipitation, the eluted proteins were analysed by western blot using a rabbit anti-MIB2 polyclonal antibody. MIB2 is immunoprecipitated by CYLD and not IgG control (panel i). Tubulin expression was used as a loading control (panel ii).