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. 2021 Jan 20;40(7):e105846. doi: 10.15252/embj.2020105846

Figure EV3. MIC19 bears both N‐ and C‐degrons.

Figure EV3

  1. Live‐cell images of 293T cells stably expressing GFP or GFP N‐terminally tagged with the wild‐type or mutant N‐terminal motif of MIC19 (aa. 1–24). Scale bar = 20 µm.
  2. Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2 or DNCul2 treatments. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red.
  3. Abundance analysis of MIC19NT‐GFP in cells treated with multiple shRNAs against ZYG11B, ZER1 or both. ZYG11B and ZER1 are BC‐box proteins involved in Gly/N‐degron recognition (Timms et al, 2019).
  4. Abundance analysis of MIC19NT‐GFP fusion proteins with (right) or without (left) a N‐terminal ubiquitin. The ubiquitin fusion technique is commonly used to characterize N‐degrons. Proteolytic cleavage of the ubiquitin moiety by Dubs resulted in exposure of MIC19NT.
  5. Live‐cell images of MIC19NT‐GFP in 293T cells with or without DNCul2 and IMP‐1088 treatments. IMP‐1088 treatment led to aggregations of 293T cells. Scale bar = 20 µm.

Source data are available online for this figure.