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. 2017 Jun 15;292(37):15254–15265. doi: 10.1074/jbc.M117.778530

Figure 5.

Figure 5.

SCCRO promotes ubiquitination of Aurora B. A, Western blot analysis of lysates from MEFs after treatment with cycloheximide at 100 μg/ml for the indicated times, showing a more rapid clearance of Aurora B in SCCRO+/+ MEFs than in SCCRO−/− MEFs. IB, immunoblot. B, Western blot analysis of lysates from MEFs after treatment with MG132 at 25 μm for the indicated times, showing an increase in the levels of Aurora B with time in SCCRO+/+ MEFs compared with SCCRO−/− MEFs (first panel). A similar increase was seen with the expression of HA-SCCRO (lanes 7–9) but not with HA-SCCROD241N (lanes 10–12). The same lysates were also subjected to immunoprecipitation using anti-Aurora B antibody and probed for polyubiquitin chains, showing enrichment of ubiquitinated Aurora B in SCCRO+/+ MEFs and HA-SCCRO–transfected SCCRO−/− MEFs but not in SCCRO−/− MEFs or HA-SCCROD241N–transfected cells (second panel). C, Western blot analysis of lysates from serum-starved (S), mimosine-arrested (M), double thymidine–blocked (T), and nocodazole-arrested (N) MEFs, showing a defect in degradation of Aurora B from M to G1 phase in SCCRO−/− MEFs compared with SCCRO+/+ MEFs. D, Western blot analysis of lysates from MEFs released from nocodazole treatment (100 ng/ml for 12 h), showing an increase of neddylated Cul3 and corresponding degradation of Aurora B in SCCRO+/+ MEFs but not in SCCRO−/− MEFs. E, the degradation of Aurora B seen in SCCRO+/+ MEFs can be reversed by addition of MG132 (50 μm) into medium at the time of release. F, the defect in Aurora B degradation observed in SCCRO−/− MEFs was rescued by retroviral introduction of HA-SCCRO but not HA-SCCROD241N. G, nocodazole-treated SCCRO+/+ MEFs released into medium containing 1 μm MLN4924 exhibited a similar defect in Aurora B degradation as that seen in SCCRO−/− MEFs. H, Western blot analysis of lysate from MEFs released from nocodazole arrest, showing a defect in degradation of Aurora B in SCCRO+/+ MEFs with KLHL21 knockdown compared with SCCRO+/+ MEFs with KLHL9 knockdown. I, duration of T2 in SCCRO−/− MEFs in the presence of either DMSO or ZM447439. DMSO or ZM447439 was added to the culture medium when cells reached the midbody stage (n = 40 for both DMSO and ZM447439 treatment, p < 0.01).