Figure 5. Rab35 regulates NDP52 translocation to damaged mitochondria.

• A, B
  Confocal micrographs of NDP52 in HeLa cells transfected with EmGFP‐Parkin and mCherry‐NDP52, exposed to 10 μM CCCP for 4 or 8 h, and immunostained with antibodies against mitochondrial TOM20 (A) or Rab35 (B). Scale bars, 10 μm.
• C
  Immunoelectron micrograph of HeLa cells transfected with EmGFP‐Rab35 and mCherry‐Parkin, exposed to 10 μM CCCP for 3 h, and labeled with anti‐GFP antibodies followed by gold particles. Scale bars, 500 nm.
• D–F
  Representative confocal micrographs (D), Pearson's coefficient between NDP52 and TOM20 (E), and NDP52 recruitment to damaged mitochondria (F) in wild‐type and Rab35 knockout cells transfected with EmGFP‐Parkin and mCherry‐NDP52, treated with 10 μM CCCP for 8 h, and immunostained with anti‐TOM20. Scale bars, 10 μm. Data in (E) are mean ± SEM from > 10 cells, and data in (F) are mean ± SEM from > 100 cells in three independent experiments.
• G, H
  Subcellular fractionation of endogenous Rab35 and NDP52. HeLa cells expressing EmGFP‐Parkin were treated with 10 μM CCCP or DMSO and fractionated. I, C, and M in Western blot images indicate input, cytosol‐rich supernatant, and mitochondria‐rich membrane pellet, respectively (G). Rab35 and NDP52 intensities were normalized to TOM20 in the mitochondria‐rich membrane pellet and quantified (H). Data in (H) are mean ± SEM from three independent experiments.

Data information: Data were tested by two‐tailed Student's t‐test: *P < 0.05, ***P < 0.001, and NS for not significant. Source data are available online for this figure.