Ubiquitin E3 ligase activity and binding to caspases of the XIAP^G466X mutant. (A) 293T cells transfected with XIAP or XIAP^G466X in the presence of ubiquitin (Ubi) or not (−) were analyzed by immunoblotting with anti-XIAP antibodies. Higher-mass products correspond to ubiquitinated XIAP species (XIAP-Ubi_n). (B) In vitro XIAP autoubiquitination assay. GST recombinant E3 ubiquitin ligase proteins (E3) were tested in the presence of ubiquitin (Ubi) and E2 and E1 ubiquitin ligases and then submitted to Western blot with anti-ubiquitin antibodies (top panels). The amount of GST proteins was controlled by Coomassie blue staining (bottom panels). (Left) Test with GST-XIAP, -XIAP^G437X, and -TRAF6. (Right) Test with GST-XIAP, -XIAP^G437X, and -XIAP^G466X. (C and D) Binding assays of XIAP and XIAP^G466X to caspases 3 and 9. (C) Glutathione-sepharose–immobilized GST-XIAP, GST-XIAP^G466X, and GST alone were incubated with different amounts of protein from dATP and cytochrome C–treated 293T cell extracts. Bound cleaved caspase 3 was detected by immunoblotting (top panel). The amount of GST proteins was controlled by Coomassie blue staining (bottom panel). (D) Different amounts of proteins extracted from 293T cells transfected with Myc-XIAP or -XIAP^G466X were immunoprecipitated (intraperitoneal) with a biotinylated peptide that corresponded to caspase 9 (CASP9) or a control peptide (Ctr.). Because the expression level of Myc-XIAP^G466X was 4- to 5-fold less expressed than Myc-XIAP (lysates, right panel), 4-fold greater amounts of proteins were immunoprecipitated from extracts that expressed Myc-XIAP^G466X.