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. 2004 Dec 23;102(1):117–122. doi: 10.1073/pnas.0405989102

Fig. 5.

Fig. 5.

Isolation and identification of S-nitrosoproteins in endothelial cells. HAECs were treated with 2 mM DEANONOate for 10 min and labeled as described in Fig. 2 except that MTSEA-biotin-X was used in place of MTSEA-Texas red. Labeled proteins were then isolated with avidin D agarose affinity chromatography. (A) Silver-stained SDS gel of S-nitrosoproteins: lanes 1 and 2, total protein of 3 μg from control and treated HAEC, respectively; lanes 3 and 4, S-nitrosoproteins isolated from 21 μg of protein of control and treated HAEC, respectively. (B) Coomassie blue stained two-dimensional gel of S-nitrosoproteins with protein spots corresponding to known proteins as determined by mass spectrometry indicated with labels. (C) Western blotting of S-nitrosoproteins from HAEC; after SDS/PAGE, proteins were transferred to poly(vinylidene difluoride) membranes and detected with a BM Chemiluminescence Blotting kit: lanes 1 and 2, total protein of 3 μg from NO-treated and control HAEC, respectively; lanes 3 and 4, S-nitrosoproteins isolated from 60μg of protein of NO-treated and control HAEC, respectively.