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. 2010 Dec 13;191(6):1141–1158. doi: 10.1083/jcb.201007152

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© 2010 Otera et al.

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Figure 3. — Mff interacts with Drp1 both in vivo and in vitro. (A) HeLa cells stably expressing FLAG-Drp1 were transfected with the indicated siRNA and further transfected with phFis1-IRES-GFP-NLS (b) and pMff-IRES-GFP-NLS (c and d). After 24 h, the cells were fixed and immunostained with anti-FLAG antibodies (red). (B) HeLa cells stably expressing FLAG-Drp1 were transfected with HA-Mff. After 24 h, the cells were immunostained with anti-HA antibodies (green) and anti-FLAG antibodies (red). High magnification images (insets) show colocalization of FLAG-Drp1 and HA-Mff. (C) HeLa cells were transfected with Mff siRNA and further transfected with FLAG-Mff-CAAX. The cells were immunostained with antibodies against Drp1 (green) and FLAG (red). Asterisks show FLAG-Mff-CAAX–nonexpressing cells. The insets show magnified images of the squared regions. (D) HeLa cells expressing the indicated proteins were treated with DSP, solubilized either with digitonin buffer (mild condition) or with SDS buffer (stringent condition), and subjected to immunoprecipitation (IP) using anti-FLAG antibody (see Materials and methods). Asterisk shows IgG light chain. IB, immunoblot. (E) Purified proteins were mixed in the indicated combinations and treated with 1 mM DSP. The reaction mixtures were solubilized with 1% Triton X-100 and subjected to immunoprecipitation with anti-Drp1 antibodies. See Materials and methods for details. (F) pMff-IRES-GFP-NLS was cotransfected with HA-WT-Drp1 or HA-Drp1-A395D into HeLa cells, and the cells were analyzed by immunofluorescence microscopy using anti-HA antibodies (red). Note that HA-WT-Drp1, but not HA-Drp1-G395A, was targeted to mitochondria in the Mff-expressing cells. (G) The cells (n = 200) for HA-WT-Drp1 and HA-Drp1-A395D with the cytosol- or mitochondria-localized pattern of Drp1 in A were quantified. vec, vector. (H) The cells treated as in F were analyzed by immunofluorescence microscopy using anti-Tom22. (I) Percentages of cells (n = 200) for HA-WT-Drp1, HA-Drp1-A395D, and HA-Drp1-K38A with the indicated mitochondrial morphology in H. Data were collected from three independent experiments and represent mean ± SD.