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. Author manuscript; available in PMC: 2011 Jul 30.
Published in final edited form as: Mol Cell. 2010 Jul 30;39(2):184–195. doi: 10.1016/j.molcel.2010.07.002

Figure 4. Regulation of XIAP E3 Ligase Activity by S-Nitrosylation in Vitro and in Intact Cells.

Figure 4

(A) In vitro S-nitrosylation of XIAP downregulates auto-ubiquitination. Purified recombinant GST-XIAP was incubated with SNOC (200 µM) or old SNOC, and 30 min later subjected to in vitro ubiquitination at RT. All samples were incubated with ATP. SNOC decreased XIAP auto-ubiquitination, as detected by anti-XIAP antibody.

(B) S-Nitrosylation of XIAP reduces auto-ubiquitination in HEK293T cells. HEK 293T cells co-transfected with HA-tagged ubiquitin plus Myc-tagged XIAP or Myc-tagged XIAPΔRING were incubated with SNOC to S-nitrosylate XIAP. After 2 to 12 hr, cell lysates were subjected to immunoprecipitation with anti-myc followed by immunoblot analysis with anti-HA to detect ubiquitinated proteins.

(C, D) S-Nitrosylation of XIAP in vitro down-regulates caspase-3 and -9 ubiquitination. Recombinant GST-XIAP was incubated with SNOC or old SNOC and then subjected to in vitro ubiquitination for 90 min at RT. All samples were incubated with ubiquitin and ATP. High molecular weight forms of caspases-3 and -9, apparently representing polyubiquitinated proteins, were detected with specific antibodies against these caspases.

(E) S-Nitrosylation of XIAP reduces caspase-3 ubiquitination in HEK293T cells. HEK293T cells co-transfected with HA-tagged ubiquitin, Fas, Myc-tagged mutant caspase-3(C285A), and His-tagged XIAP were exposed to SNOC. After 6 hr, cell lysates were immunoprecipitated with anti-myc antibody followed by immunoblot analysis with the indicated antibodies. Arrowhead, procaspase-3; *, immunoglobulin light chain.

(F) S-Nitrosylation of XIAP reduces caspase-9 ubiquitination in HEK293T cells. HEK 293T cells co-transfected with HA-tagged ubiquitin, Bax, FLAG-tagged mutant caspase-9(C285A), and Myc-tagged XIAP were exposed to SNOC. After 6 hr, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblot analysis with the indicated antibodies. Arrowhead, XIAP; *, immunoglobulin heavy chain.