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. Author manuscript; available in PMC: 2018 May 11.
Published in final edited form as: Nat Chem Biol. 2017 Oct 9;13(12):1239–1244. doi: 10.1038/nchembio.2493

Figure 5. Stendomycin inhibits mitochondrial inner-membrane translocation but does not stabilize PINK1 or trigger mitophagy.

Figure 5

(a) HeLa cells expressing GFP-Parkin were transfected with control (scr, scrambled) or PINK1 siRNAs in the presence of DMSO (0.1%), stendomycin (stendo), CCCP (10 μM) or a combination of oligomycin (100 μM) and antimycin A (100 μM) (OA) for 4 or 20 h. Total cell lysates were analyzed by western blotting as indicated. Similar data was observed in one independent repeat for all markers shown. P-Ub, phospho Ser65-ubiquitin; PINK-FL, full-length PINK1. (b) In vitro import assays were performed as in Figure 4c using PINK1 as substrate. p, precursor; c, PARL-cleaved PINK1. (c) Radiolabeled human PINK1 was in vitro imported into isolated HeLa cell mitochondria in the presence or absence of 500 nM stendomycin for 5 and 20 min. The membrane potential was disrupted in indicated reaction by the addition of 25 μM CCCP. 1% DMSO served as the vehicle control. The membrane insertion of PINK1 was analyzed by alkali extraction for all reactions, which was followed by SDS-PAGE and autoradiography. (d) As in c but with mitochondria solubilized in digitonin and separated by BN-PAGE. PINK1 complexes were visualized by autoradiography. Full gel scans are in Supplementary Information.