cIAP1/2 Are Direct E3 Ligases Conjugating Diverse Types of Ubiquitin Chains to Receptor Interacting Proteins Kinases 1 to 4 (RIP1–4)
Figure 1
XIAP, cIAP1 and cIAP2 bind RIP1-4 proteins.
(A) In vitro-transcribed and -translated RIP1, 2, 3 and 4 labeled with 35S-methionine (Input) was incubated overnight with bacterially produced GST, GST-XIAP, GST-cIAP1, or GST-cIAP2 bound to Sepharose beads. The beads were washed extensively and run on an acrylamide gel. Binding of RIPs was revealed by autoradiography (pull-down). (B) In vitro-transcribed and -translated RIP1, 2, 3 and 4 labeled with 35S-methionine (Input) was incubated overnight with bacterially produced GST-XIAP, GST-cIAP1, or GST-cIAP2 bound to Sepharose beads in presence of BV6 (5 µM) or DMSO as a control. The beads were washed extensively and run on an acrylamide gel. Binding of RIPs was revealed by autoradiography. (C) HEK293T cells were transfected with VSV-tagged RIP plasmids in the absence or presence of a FLAG-tagged cIAP1 plasmid. cIAP1 was immunoprecipitated in NP-40 buffer using anti-FLAG antibody and coimmunoprecipitated RIPs were revealed by immunoblotting with anti-VSV antibody. Protein expression in the lysates is shown. (D) HEK293T cells were transfected with VSV-tagged RIP plasmids in the absence or presence of a Myc-tagged cIAP2 plasmid. cIAP2 was immunoprecipitated in NP-40 buffer using anti-Myc antibody and coimmunoprecipitated RIPs were revealed by immunoblotting with anti-VSV antibody. Protein expression in the lysates is shown.