cIAP1-CARD-RING mediated degradation of XIAP and Livin, but not cIAP1 and cIAP2, occurs independently of E1. (A) HeLa cells were transfected with nontargeting luciferase siRNA (NT) or ubiquitin-activating enzyme-specific siRNA (E1) for the indicated times. Protein extracts were subjected to Western immunoblot analysis with antibodies against ubiquitin, E1, and GAPDH. (B–E) After 80 h of siRNA transfection, HeLa cells were transfected with 6myc-cIAP1 (B), cIAP2 (C), XIAP (D), or Livin (E) in the presence or absence of LacZ, cIAP1-CR, cIAP1-CR-H588A, and Xenopus E1 for an additional 24 h. Protein extracts were collected and subjected to Western immunoblot analysis. 6myc-proteins, cIAP1-CR, and Xenopus E1 were detected with anti-myc, anti-FLAG, and anti-V5 antibodies, respectively.