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. 2010 Apr 7;5(4):e10054. doi: 10.1371/journal.pone.0010054

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Poole et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Affinity purified anti-dMfn antiserum was used to immunoprecipitate dMfn from wild-type flies, PINK1^B9 mutants, and park²⁵ mutants. As a control for specificity, anti-dMfn antiserum was also used to immunoprecipitate dMfn from flies with hsp70-GAL4 driven expression of UAS-dmfn-RNAi^Vienna. In the left panel, 3% of the lysate input used in the immunoprecipitations was subjected to western blot analysis using an anti-ubiquitin antiserum to show that general ubiquitination levels were similar in all genotypes. In the middle and right panels, dMfn immunoprecipitates from all four genotypes were subjected to western blot analysis with either anti-ubiquitin antiserum (middle panel) or anti-dMfn antiserum (right panel). Arrow indicates the unmodified dMfn species detected in wt flies, PINK1^B9 mutants, and park²⁵ mutants, with reduced levels in flies expressing UAS-dmfn-RNAi^Vienna. Arrowhead indicates location of ubiquitinated dMfn species. These analyses were replicated at least three times with similar results.