Fig. 1.
Effect of diamide on hRyR2WTcross-linking and roGFP-PLB oxidation. A, Western blot images of hRyR2 cross-linking induced by increasing concentrations of diamide. The control samples were treated with 5 mM DTT to prevent hRyR2 oxidation and cross-linking. B, a schematic diagram of the roGFP-PLB sensor. The redox sensor roGFP was fused to the N-terminus of PLB to measure oxidative stress at the cytosolic side of ER membrane (left). Expression pattern of the roGFP-PLB sensor in HEK293 cells (right). C, changes of the roGFP-PLB signal in response to increasing concentrations of diamide. As the roGFP-PLB signal decreases with an increase of roGFP oxidation, the recorded signal (F) was presented as background-subtracted (1−F/FMax) values. The background fluorescence was recorded during the maximum roGFP oxidation with 100 μM diamide. FMax was recorded in the presence of 5 mM DTT. D, dose-dependence of the hRyR2WT cross-linking (n = 5 western blots) and the roGFP-PLB signal (n = 11 cells) from diamide concentration. The data were fitted by the Hill equation using the built-in Origin 2016 SR Hill function, which was also used to determine the Michaelis constant (Km).