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. 2018 Jan 17;37(5):e98308. doi: 10.15252/embj.201798308

Figure EV3. p62‐dependent cluster formation is triggered by poly‐ubiquitinated proteins.

Figure EV3

  1. Representative images of cluster formation assays conducted with 5 μM GST‐4xUb and the indicated mCherry‐p62 concentrations. Images were taken before the addition of GST‐4xUb or 60 min after. Representative images of three independent replicates.
  2. Quantification of p62 cluster formation in the presence of the indicated free ubiquitin chains. GST‐4xUb was used as a positive control. Averages and SDs from three independent replicates are shown. Samples were imaged for 15 min and then again after 45 min. Dashed lines represent the extrapolated curves for a full time lapse. For GST‐4xUb data points from the peak of the curve to the latest time point (t = 45 min) were fitted to a single exponential decay (R 2 = 0.9973). Box: representative micrographs of the indicated samples 45 min after addition of Ub chains. Pictures are displayed in false colors (ImageJ: fire).
  3. Left: Coomassie Brilliant Blue‐stained gel showing the assembly of GST‐tagged K48‐ and K63‐linked ubiquitin chains. Sup.: supernatant. Right: purified GST‐tagged ubiquitin chains were subjected to Western blotting with the indicated antibodies.
  4. Left: quantification of aggregation assays conducted with 2 μM mCherry‐p62 and 20 μM GST‐4xUb in the presence or absence of 200 μM GFP‐Ub, GFP, or Ub. Averages and SDs from four independent experiments are shown.
  5. mCherry‐2xFKBP‐p62 was subjected to size‐exclusion chromatography in the presence or absence of the homodimerizer AP20187. Fractions were collected and run on a SDS–PAGE gel. The corresponding elution volume is indicated for each fraction.
  6. Cluster formation assays conducted with mCherry‐p62 WT or ‐2xFKBP in the presence or absence of GST‐4xUb and AP20187. Average and SDs of three independent experiments are shown.