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. 2012 Aug 21;3:1016. doi: 10.1038/ncomms2016

Figure 1. PINK1 is phosphorylated following a decrease in mitochondrial ΔΨm.

Figure 1

(a) Endogenous PINK1 in the presence of CCCP (10 μM, 1 h) recruits Parkin to mitochondria more efficiently than over-produced PINK1 (CMV promoter-driven) in the absence of CCCP. The number of cells with Parkin-positive mitochondria in the Parkin-expressing cells was counted in >100 cells. Error bars represent the mean±s.d. values of three experiments. Statistical significance was calculated using analysis of variance with a Tukey–Kramer post hoc test. Example figures indicative of colocalization and no colocalization are shown on the right (scale bars, 10 μm). (b) Immunoblotting of exogenous and endogenous PINK1. The asterisk indicates the position of endogenous PINK1. Actin was used as a loading control. FL, full-length PINK1; Δ1, the N-terminal processed form. (c) HeLa cells expressing non-tagged PINK1 were treated with 10 μM CCCP for the indicated times and subjected to SDS–PAGE on a 7.5% Tris-glycine gel, a 4–12% precast gel or a 50 μM phos-tag containing gel. Red asterisks show phosphorylated PINK1. We routinely used HeLa cells and immunoblotting analysis was conducted with an anti-PINK1 antibody unless otherwise specified. (d) In vitro synthesized PINK1-3HA and the mitochondrial fraction of PINK1-3HA-expressing cells following CCCP treatment were subjected to immunoblotting. (e) The cell lysate and immunoprecipitated product of PINK1−/− MEFs-expressing WT or the PINK1 KD mutant were subjected to SDS–PAGE on a 7.5% Tris-glycine gel ±50 μM phos-tag. Red asterisks show phosphorylated PINK1. (f) The endogenous PINK1 in HeLa cells exists as the phosphorylated form. The black asterisk indicates a cross-reacting band and red asterisks show phosphorylated PINK1. (g) Phosphatase treatment caused the high-molecular shift of both exogenous and endogenous PINK1 to disappear. The mitochondrial fraction collected from HeLa cells-expressing PINK1-HA or collected from noninfected HeLa cells was treated with CIAP at the indicated temperature. Exogenous PINK1 was detected with an anti-HA antibody. + and ++ mean 10 and 30 U per reaction of CIAP, respectively. (h) Cells expressing exogenous PINK1 were treated with CCCP (10 μM, 1 h), valinomycin (10 μM, 1 h) or rotenone (200 μM, 24 h) and subjected to SDS–PAGE on a 7.5% Tris-glycine gel ±50 μM phos-tag.