Figure 3. Combined effects of PKA phosphorylation and oxidation on calstabin2 binding to RyR2.

(A) CSR preparations were treated with 1 mM H₂O₂, and RyR2 was immunoprecipitated, size fractionated, and immunoblotted for oxidation (DNP) and calstabin2 in the RyR2 complex. (B) Levels of oxidation and calstabin2 in the RyR2 complex were normalized to the total amount of RyR2 (AU). *P < 0.05 untreated versus H₂O₂-treated samples. (C) GSH/GSSG ratios (n = 2) were compared between WT and RyR2-S2808D^+/+ mice. *P < 0.05. (D) ³⁵S-calstabin binding was measured in samples treated with PKA, H₂O₂, or a combination of the 2, in the presence or absence or S107. Radioactivity counts were normalized to the untreated control samples. Data are presented as mean ± SEM (n = 4). The numbers of replicates for each condition are indicated by the parenthetical numbers over each bar. *P < 0.05 compared with control; ^#P < 0.05 compared with PKA/H₂O₂ treatment without S107. (E) CSR was treated with 1 mM H₂O₂, and RyR2 was immunoprecipitated and immunoblotted for oxidation (DNP) and PDE4D3 in the RyR2 complex. (F) RyR2 was expressed in CHO cells using an inducible vector (tetracycline), transient transfection, or stable transfection and immunoblotted with anti-DNP antibody to determine oxidation of the channel.