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. 2014 Dec 19;290(5):2798–2811. doi: 10.1074/jbc.M114.620906

FIGURE 7.

FIGURE 7.

Thr-313 and Ser-402 are important for Parkin recruitment by PINK1. A, WT (PINK1+/+) and PINK1 KO (PINK1−/−) HeLa cells were treated with 10 μm CCCP for 3 h and analyzed for PINK1 expression via Western blot (WB) analysis. No endogenous PINK1 was detected in PINK1−/− cells. B, HeLa cells were transfected with Parkin-GFP and treated with CCCP for 3 h. Immunohistochemistry for Parkin-GFP (green) and the mitochondrial marker cytochrome c (red) shows that WT (PINK1+/+) but not PINK1 KO (PINK1−/−) HeLa cells display mitochondrial Parkin recruitment. Expression of WT PINK1 in PINK1 KO cells rescues Parkin recruitment. Immunoblot analysis using anti-β-actin shows equal loading. C, quantification of Parkin recruitment in WT (PINK1+/+), KO (PINK1−/−), and KO HeLa cells rescued with human WT PINK1 after 3 h of DMSO or 10 μm CCCP treatment. Statistical significance was calculated between HeLa WT and PINK1 KO rescued cells using Student's t test. Data are mean ± S.E. (n = 6 independent experiments). D, PINK1 KO HeLa cells were transiently transfected with WT and mutant PINK1 and subsequently treated with DMSO or 10 μm CCCP for 3 h. PINK1 expression levels were analyzed by Western blot analysis. Immunoblot analysis using anti-β-actin shows equal loading. E, quantification of Parkin recruitment in PINK1 KO HeLa cells rescued with human WT or mutant PINK1 after 3 h of 10 μm CCCP treatment. Statistical significance was calculated between each mutant and WT PINK1 using Dunnett's test. **, p <0.01; ***, p < 0.001; ns, nonspecific. Data are mean ± S.E. (n = 4 independent experiments).