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. 2014 Nov 19;290(1):296–309. doi: 10.1074/jbc.M114.560169

FIGURE 5.

FIGURE 5.

Binding of ubiquitinated proteins affects SCCRO-promoted nuclear translocation of cullins. A, immunoblot (IB) analysis of lysates from U2OS cells transfected with the indicated constructs following immunoprecipitation (IP) for MYC showing no differences between the binding of SCCRO and SCCROL30A to neddylation components. B, Western blot analysis of the pulldown products of GST-SCCRO and SCCRO mutants from HeLa cell extracts probed with the indicated antibodies showing that SCCRO binds to CAND1, CUL1, and ROC1. The cullin-binding mutant SCCROΔ210–259 loses its ability to bind to CAND1, CUL1, and ROC1; in contrast, UBA mutants do not. C, immunofluorescence analysis using rhodamine-conjugated anti-MYC or anti-V5 and FITC-conjugated anti-HA on U2OS cells transfected with MYC-SCCRO, MYC-SCCROL30A, V5-SCCRO3KR, or MYC-NLS-SCCRO and HA-CUL1Δ610–615 in the presence or absence of MG132. Pretreatment with MG132 caused translocation of a significant proportion of MYC-SCCRO, but not MYC-SCCROL30A, V5-SCCRO3KR, or MYC-NLS-SCCRO, from the nucleus to the cytoplasm (first row). In addition, pretreatment with MG132 blocked nuclear translocation of HA-CUL1Δ610–615 promoted by SCCRO but not by SCCROL30A, V5-SCCRO3KR, or MYC-NLS-SCCRO (second row). D, fractionation analysis of U2OS cells transfected with HA-SCCRO showing increased levels of cytosolic SCCRO and monoubiquitinated SCCRO but not SCCROL30A after treatment with MG132. Results from densitometry measurement of Western blots from three independent experiments are shown (C, cytoplasm; N, nucleus; C/N stands for ratios of the levels of cytosolic SCCRO or SCCROL30A versus the levels of nuclear SCCRO or SCCROL30A). *, p = 0.043. E, Western blot analysis of neddylated CUL1 and CUL3 in U2OS cells transfected as indicated showing that expression of NLS-SCCRO or SCCRO3KR resulted in increased levels of neddylated CUL1 and CUL3 to an extent similar to expression of SCCRO. Western blotting for SCCRO on the same lysates showed that although both MYC-SCCRO and MYC-NLS-SCCRO underwent monoubiquitination V5-SCCRO3KR did not under identical conditions (1, monoubiquitinated SCCRO; 2, endogenous SCCRO). PCAF, p300/CBP-associated factor; EV, empty vector.