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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 3. Identification of the Predominant S-Nitrosylation Site on XIAP and Formation of SNO-XIAP in Vivo.

Figure 3

(A) S-Nitrosylation of XIAP at Cys450 by NO-biotin switch assay. HEK-nNOS cells were transfected with WT, C450H, or C471H XIAP constructs and exposed to the Ca2+ ionophore A23187 (5 µM) to activate endogenous nNOS.

(B) ETD-MS/MS spectra of XIAP-RING domain. To obtain MS/MS spectra, charge +6 precursors ([M+6H]+6) of unmodified RING (RING, top; 1053.68 m/z) and, after exposure to 10 µM SNOC, S-nitrosylated RING (SNO-RING, bottom; 1058.40 m/z) were isolated for the ETD experiment by a linear ion trap (LTQ) analyzer. Amino-acid sequence of the XIAP-RING domain is listed at the top of the RING spectrum. c and z ions present in both modified and unmodified proteins are indicated in blue, while ions detected only in the SNO-RING are in red. m/z values for C11+3,C13+2, and C16+3 ions in the SNO-RING spectrum are 425.17, 755.36, and 632.17, respectively. Additional MS data (LTQ Orbitrap XL-MS and ETD-MS/MS) are available in Figures S3A and S3B.

(C) SNO-XIAP in brains of patients with neurodegenerative diseases. Postmortem brain tissues from patients with neurodegenerative and non-CNS conditions (controls) were subjected to the NO-biotin switch assay. Images separated by a black line are from the same gel.