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. Author manuscript; available in PMC: 2012 Nov 11.
Published in final edited form as: Cell. 2011 Nov 11;147(4):893–906. doi: 10.1016/j.cell.2011.10.018

Figure 5. PINK1 and Parkin Interact with Miro upon Mitochondrial Depolarization.

Figure 5

The interactions of PINK1, Parkin, and Miro were examined in HEK293T cells transfected as indicated. For each assay, 50% of the precipitate and 10% of the input were loaded. (A, B) Immunoprecipitations using anti-Myc, anti-Flag or anti-GFP. PINK1-Flag (A) and YFP-Parkin (B) were detected in Myc-Miro immunoprecipitates, and Myc-Miro was detected in PINK1-Flag (A) or YFP-Parkin immunoprecipitates (B). MG132 was used to prevent Myc-Miro degradation. (C, D) Immunoprecipitation of endogenous PINK1 (C) or Parkin (D). Myc-Miro coprecipitated efficiently with anti-PINK1 or anti-Parkin when cells were treated with 40µM CCCP for 10 min. (E, F) HEK293T cells transfected with Myc-Miro were incubated with 10µM CCCP in DMSO or DMSO alone for 3 h prior to lysing the cells. Immunoblots of lysates were probed with anti-Myc and detected with a fluorescence scanner for quantification (F) after normalization to the mitochondrial loading control ATP5β and expressed as a fraction of the control value. n=6 transfections. (G) Cell lysates from HeLa cells transfected as indicated and exposed to CCCP or DMSO as in (E). (H) Quantification of Myc-Miro levels, normalized to ATP5β and expressed as a fraction of the DMSO control. n=4 transfections. See also Figure S4.