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. 2010 Dec 27;191(7):1367–1380. doi: 10.1083/jcb.201007013

Figure 4.

Figure 4.

Mitofusin ubiquitination requires Parkin and depolarization of mitochondria. (a) Mitochondrial fractions (30 µg) from HeLa cells treated with or without CCCP and recombinant MBP-Parkin wild-type or MBP-Parkin T415N proteins (left) were subjected to the in vitro ubiquitination assay (right). Isolated mitochondria were incubated with MBP-Parkin wild type or T415N. Reaction mixtures were subjected to the denatured IP with anti-Mfn1 antibody as in Fig. 2. IP products were detected with anti-Mfn1 and anti-Ub (P4D1). UbcH7 was used as an E2 enzyme. The asterisk indicates a nonspecific band from MBP proteins or other reaction components. (b) HeLa cells or HeLa cells stably transfected with YFP-Parkin were subjected to nondenaturing IP with anti-Parkin antibody (PRK8). IP products were detected by anti-Parkin, Mfn1, and Mfn2. CCCP, 10 µM for 90 min; MG132, 30 µM, 30 min prior and with CCCP. An asterisk on the Mfn2 panel indicates a nonspecific band and the double asterisks indicate a cross-reactive band to Mfn1. Molecular mass is indicated in kilodaltons next to the gel blots.