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. 2018 Jul 4;37(16):e98701. doi: 10.15252/embj.201798701

Figure 1. Cezanne is a cell cycle‐regulated, K11 linkage‐specific DUB .

Figure 1

  1. Recombinant GST‐Cezanne (0.2 μM) was incubated with 1 μM of the indicated diubiquitin probes in DUB reaction buffer at room temperature. Aliquots were collected at the indicated time points and analyzed by silver stain.
  2. Recombinant GST‐Cezanne (0.1 μM) was incubated with 1 μM of K11‐linked DiUb or TetraUb in DUB reaction buffer at room temperature. Aliquots were collected at the indicated time points and analyzed by silver stain.
  3. U2OS cells were synchronized in mitosis with nocodazole, isolated by “shake‐off”, and analyzed by immunoblot after release into the cell cycle.
  4. HCT116 cells grown asynchronously or synchronized in mitosis with nocodazole and isolated by “shake‐off” were analyzed by immunoblot with the indicated antibodies.
  5. Representative immunofluorescence images stained for Cezanne, Tubulin, and DNA during the cell cycle in U2OS. Quantification of Cezanne intensity between interphase and mitotic cells is shown on the right (error bars show standard deviation for n = 105 and 28 interphase and mitotic cells, respectively).