Figure 2
These changes in SNO signalling are quantifiable using a novel split-renilla based luciferase biosensor. A split renilla biosensor was created based on the SNO-dependent interaction of Xpo1 and REV as described previously17 (A) Each half of renilla (8.1 and 8.2) was cloned onto the C-terminus of each protein. (B) When a critical cysteine in Xpo1 is nitrosated, the Xpo1-REV interaction is abolished. (NES = nuclear export signal). (C) Overexpression of the individual proteins, lagged with Rluc8.1 were detected using Western blotting using specific antibodies, and (D) Co-overexpression of Xpo1–8.1 and REV-8.2 results in a luminescence signal, as detected using a luminescent plate reader, which is significantly greater than that of Xpo1/GST and REV/GST negative control overexpression. GST/GST = positive control. (E) This SNO-dependent interaction of XPO1 and REV responds to a dose response of GSNO and LMB. A decreased signal is observed in the presence of either GSNO (0.5–2 mM) or LMB (2.5–15 ng/ml). When these constructs were expressed in either (F) sEnd.1 endothelial cells in the presence or absence of GCH1-specific siRNA, or (G) eNOS/GCH tet-regulatable cells plus doxycycline, an increased interaction was detected indicative of decreased cellular SNO levels. (n = 5/6, *P < 0.05).
