FIGURE 3.

Thr-313 and Ser-402 are required for PINK1 phosphorylation at the mitochondrial outer membrane. A, mitochondrially enriched fractions from HEK cells transiently transfected with different phosphomutant forms of PINK1-FLAG and treated with DMSO or 10 μm CCCP were analyzed by SDS-PAGE on 7.5% Tris acetate and 7.5% Phos-Tag gels. The presence of P-FL, FL, and ΔMTS PINK1 was assessed by immunoblotting using anti-FLAGM2 antibody. Although FL PINK1 accumulated upon CCCP treatment for every evaluated mutant, P-FL PINK1 was not detected or altered for T313A, S402A, and the 4×A quadruple mutant PINK1. WB, Western blot. B, mitochondrial fractions from CCCP-treated HEK cells transfected with different phosphomutant PINK1-FLAG forms were treated with LPP and further probed with anti-FLAGM2 antibody for PINK1 detection. The bands P-FL(A) and P-FL(B) show sensitivity to LPP, indicating that they are both phosphorylated forms of PINK1 (P-FL A and B).