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. Author manuscript; available in PMC: 2012 Jan 7.
Published in final edited form as: Mol Cell. 2010 Dec 23;41(1):107–116. doi: 10.1016/j.molcel.2010.12.002

Figure 5. Dominant-negative Siah alters XIAP protein levels.

Figure 5

A. Siah-1ΔRING dominant-negative blocks ARTS-induced decline in XIAP protein levels. HEK293T cells were transfected with plasmids encoding FLAG-ARTS versus empty plasmid (CNTL) together with plasmids encoding full-length (FL) Myc-Siah-1, or Myc-Siah ΔRING in the presence of the pan-caspase inhibitor z-VAD-fmk (20 μM). At various times, cell lysates were analyzed by immunoblotting. B. RING domain of Siah-1 but not RING of XIAP is required for ARTS-induced ubiquitination of XIAP. HEK293T cells were transfected with Myc-XIAP, Myc-XIAPΔRING, HA-ARTS, FLAG-Siah-1, and/or FLAG-SiahΔRING. After 24 hrs, cell lysates were normalized for protein content and IPs were performed using mouse monoclonal anti-Myc antibody. IPs and lysates were analyzed by immunoblotting. C. Siah-1 is required for ARTS-induced ubiquitination of XIAP. Wild-type (WT) or Siah1/2 −/− double knockout (KO) MEFs were transiently transfected with Myc-XIAP, HA-ARTS, and/or plasmids encoding FLAG-Siah-1, FLAG-Siah-2, or both. Lysates were IP’ed using anti-Myc. IPs and lysates were analyzed by immunoblotting.