SCCRO promotes Cul3 neddylation and is required for its localization to the midbody.
A, Western blot analysis of cell lysates, showing that neddylation of Cul3 was reduced in SCCRO−/− MEFs more than other cullins compared with SCCRO+/+ MEFs. The asterisk denotes possible dineddylated Cul5. IB, immunoblot. B, Western blot analysis of cell lysates of infected SCCRO−/− MEFs, showing that retrovirus-mediated transfection with HA-SCCRO restored SCCRO expression to levels seen in SCCRO+/+ MEFs (center row, compare lanes 2 and 4) and rescued Cul3 neddylation (lane 4). EV = empty vector. C, immunostaining for cullins and Aurora B, showing that only Cul3 localizes to the midbody in SCCRO+/+ MEFs and also showing the absence of Cul3 in the midbody of SCCRO−/− MEFs and MLN4924-treated SCCRO+/+ MEFs. Note that anti-Cul4 did not work for immunostaining. Scale bar = 5 μm.