Figure 6

Analysis of XIAP-mediated ubiquitination of XIAP and RIP2. (a) Ub conjugates were purified with Protein A or G magnetic beads from lysates of HEK 293 T cells transfected as indicated and examined by immunoblotting. RIP2 ubiquitination was defective in HEK 293 T cells for all XIAP mutants. (b) XIAP expression was analyzed in HEK 293 T cells transfected GFP-tagged XIAP WT or mutant constructs, along with RFP-tagged RIP2 by LC–MS. XIAP mutants expression levels were significantly lower compared to XIAP WT expression level. (c) XIAP ubiquitination was analyzed in HEK 293 T cells transfected with GFP-tagged XIAP WT or mutant constructs, along with RFP-tagged RIP2 by MS. Auto-ubiquitination of XIAP mutants were significantly higher compared to that of XIAP WT.