cIAP1/2 Are Direct E3 Ligases Conjugating Diverse Types of Ubiquitin Chains to Receptor Interacting Proteins Kinases 1 to 4 (RIP1–4)
Figure 3
cIAP1 conjugates RIP1–4 proteins with Lys48-, Lys63-, and linear ubiquitin chains.
(A) Schematic representation of the different ubiquitin mutants used in the experiments. (B–D) In vitro ubiquitination assays were performed on in vitro-transcribed and -translated RIP1–4 proteins labeled with 35S-methionine. GST or GST-cIAP1 was used as E3 component and UbcH5a as the E2 component. The reaction was carried out in presence of (B) wild-type (WT) ubiquitin, K48-only ubiquitin and K63-only ubiquitin; (C) no ubiquitin, wild-type (WT) ubiquitin and lysine free ubiquitin (KO); (D) no ubiquitin, wild-type (WT) ubiquitin and Myc-tagged ubiquitin. Ubiquitination of the RIPs was revealed by autoradiography.