Journal: The EMBO Journal
Article Title: Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control
Figure Lengend Snippet: Cell-wide clearance of mitochondria via autophagy is triggered by depolarization and proceeds over a 24-h time period Representative immunoblot of whole-cell lysates from U2OS:GFP and GFP-parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period. Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. ( n = 3 experiments). Quantification of mitochondrial clearance in U2OS:GFP-parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. ( n = 3 experiments, with at least 85 cells quantified per condition, per experiment). U2OS:GFP-parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single-channel images. Scale bars, 20 μm. Source data are available online for this figure.
Article Snippet: 1:1,000), anti-beclin-1 (BD Biosciences, 612113, 1:1,000), anti-Drp1 (BD, 611113, 1:5,000), anti-GAPDH (Novus Biologicals, NB300-320, 1:50,000), anti-parkin (Santa Cruz Biotechnology, sc-32282, 1:2,000 [endogenous parkin] or 1:100,000 [ectopic parkin]), anti-PINK1 (Novus, BC100-494, 1:2,500), anti-Rab9 (Abcam, ab2810, 1:1,000), anti-TIM23 (BD, 611222, 1:40,000), anti-TOM20 (Santa Cruz Biotechnology, sc-11415, 1:50,000), anti-p97/VCP (Abcam, ab11433, 1:10,000), anti-PDH E1a (Abcam, ab110330, 1:5,000), anti-TRAP1 (Abcam, ab2721, 1:5,000), anti-α-tubulin (Santa Cruz Biotechnology, sc-23948, 1:50,000) and anti-VDAC1 (Abcam, ab14734, 1:40,000).
Techniques: Western Blot, Staining, Fluorescence, Microscopy