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

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 4. — SCCRO regulates abscission through the Cul3^KLHL21 complex. A, Western blot analysis of lysates from SCCRO^+/+ MEFs treated with shRNA against KLHL9 or KLHL21 as indicated and probed with KLHL9, KLHL21, and α-tubulin antibodies. C, lacZ shRNA knockdown control; IB, immunoblot. B, KLHL9 (left panel) and KLHL21 (right panel) shRNA-treated SCCRO^+/+ MEFs stained for Aurora B (green), α-tubulin (red), and nuclei (blue). KLHL21 shRNA-treated cells show increased levels of midbody cells. Insets, close-up view of midbody cells. The percentages of midbody cells are included inside the images. Scale bar = 20 μm. C, DAPI staining, showing an increased percentage of polyploidy cells in SCCRO^+/+ MEFs with KLHL21 knockdown (right panel) compared with those with KLHL9 knockdown (left panel). Scale bar = 20 μm. D, immunofluorescence analysis using anti-pericentrin, showing supernumerary centrosomes in SCCRO^+/+ MEFs with KLHL21 knockdown but not in those with KLHL9 knockdown. Scale bar = 5 μm. E, immunostaining for Cul3 and Aurora B, showing an absence of Cul3 from the midbody in SCCRO^+/+ MEFs with KLHL21 knockdown (shKLHL21) but not in SCCRO^+/+ MEFs with KLHL9 knockdown (shKLHL9) or lacZ knockdown controls (shlacZ). Scale bar = 5 μm. F, immunostaining for KLHL21 and Aurora B, showing an absence of KLHL21 in the midbody of SCCRO^−/− MEFs and MLN4924-treated SCCRO^+/+ MEFs compared with SCCRO^+/+ MEFs (top panel). Note that there is no difference in KLHL9 localization between SCCRO^+/+ and SCCRO^−/− MEFs (bottom panel).