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. 2013 Mar 1;24(5):659–667. doi: 10.1091/mbc.E12-10-0721

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© 2013 Losón et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Overexpression of MiD49 or MiD51 causes enhanced phosphorylation of Drp1 on S637. (A) Increased Drp1 S637-PO₄ in cells overexpressing MiD49 or MiD51. Top, lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1 and the Myc-tagged MiD. Bottom, Drp1 was immunoprecipitated, and the levels of Drp1 S637-PO₄ were detected with a phosphospecific Drp1 antibody. Drp1 was used as a loading control for the immunoprecipitated samples. (B) Drp1 S616-PO₄ levels in cells overexpressing MiD49 or MiD51. Lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1, Drp1 S616-PO₄, and the Myc-tagged MiD. Actin was used as a loading control. (C) Increased Drp1 S637-PO₄ recruitment to mitochondria in cells overexpressing MiD49 or MiD51. Top, cytosol and crude mitochondrial fractions were prepared from the indicated HeLa cells and analyzed by Western blotting for Drp1, superoxide dismutase 2 (SOD2; mitochondrial), and β-tubulin (TUBB; cytosolic). The mitochondrial lanes were loaded with 20-fold more cell equivalents than with the cytosolic lanes. Bottom, the loading of mitochondrial fractions was normalized for the total Drp1 level. The relative levels of Drp1 S616-PO₄ and Drp1 S637-PO₄ on mitochondria were assessed by Western blotting with phosphospecific antibodies. In A–C, densitometry was performed on the S616-PO₄ and Drp1 S637-PO₄ blots and was normalized to the total Drp1 in each sample. Values are presented as proportions of wild type. (D) Binding of MiD49 and MiD51 to phospho-mutants of Drp1. 293T cells were cotransfected with Myc-tagged MiD and Drp1 mutants as indicated. Cells were treated with cross-linker and solubilized. Top, expression of Drp1 and Myc-MiD in the lysates. Bottom panel, anti-Myc immunoprecipitates were analyzed for Drp1. Note that this assay only detects transfected Drp1 (compare lanes 2, 3–7, and 10). A, phospho-null mutant; D, phosphomimetic mutant; S, wild-type Drp1.