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. 2021 Feb 5;7(6):eabe3706. doi: 10.1126/sciadv.abe3706

Fig. 7. Calcified human aortic valves display reduced S-nitrosylation of USP9X along with decreased expression of MIB1 and NICD.

Fig. 7

(A) Immunoblot for USP9X shows significantly reduced S-nitrosylation in aortic valves from adults with moderate (AVD-1 and AVD-2) and moderate-severe (AVD-3 and AVD-4) CAVD when compared to uncalcified aortic valve (Control). S-nitrosylation of GAPDH is also shown. Absence of a band in the ascorbate-negative experiment served as the negative control. Total load shows protein expression in the cell lysates before purification of S-nitrosylated proteins where USP9X, MIB1, and NICD expression is reduced in calcified valves while RUNX2 is increased. GAPDH serves as a loading control. (B) Schematic showing the proposed model where S-nitrosylation of USP9X leads to MIB1 stabilization via deubiquitination, which increases ligand-mediated NOTCH1 activation in neighboring cells and the inhibition of calcification in physiological concentrations of NO. This pathway is abrogated under low concentrations of NO.