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. 2020 Jun 28;19(15):1899–1916. doi: 10.1080/15384101.2020.1782036

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Figure 5. — Depletion of RanBP1 decreases RCC1 dynamics. (a) FRAP analysis of RCC1 dynamics on metaphase chromatin in HCT116^{RanBP1-μAID/HA} cells expressing RCC1-iRFP670::TIR1 and RanBP1-µAID-HA treated with (blue) or without (black) 1 mM Auxin for 3 h. (b) FLIP analysis cells, as in (A). (c) FRAP analysis of RCC1 dynamics on chromatin during anaphase B/telophase. Results are expressed as an average from a total of 12 measurements recorded from 3 independent experiments. (significance was assessed by F test). (d) RanBP1 was immunoprecipitated from mitotic lysates of control or Auxin-treated HCT116^{RanBP1-μAID/HA} cells that express RanBP1-µAID-HA and RCC1-iRFP670 using HA antibody. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against RCC1, RanBP1, Ran, HA and RanBP3. Total lysate of parental HCT116 cells is shown in the first lane, for comparison purposes. Note both efficient interaction of RCC1 and RanBP1 (lane 4) and its specificity (lane 5).