Figure 7.

Effects of compound #33 on CRL1/SCF^β-TrCP-dependent β-catenin ubiquitination. A, immunoblot confirmed extensive neddylation of CUL1. 100 ng of Nedd8-ROC1/Rbx1-CUL1 (lanes 1 and 2, top; a gift of Ning Zheng of University of Washington, Seattle) or ROC1/Rbx1-CUL1 (lanes 1 and 2, bottom; purchased from R&D Systems) was subjected to immunoblot analysis using anti-CUL1 (lane 1) or anti-Nedd8 (lane 2) antibodies. Antibody-reactive signals were visualized by Li-COR Odyssey scanner. Nedd8-ROC1/Rbx1-CUL1, but not ROC1/Rbx1-CUL1, reacted to anti-Nedd8 antibody. Signal merge in lane 3 shows the complete overlap of anti-CUL1 and anti-Nedd8 reactive signals, indicating quantitative neddylation of CUL1. B, reactions with Nedd8-CRL1/SCF^β-TrCP. Incubation is 10 min at 37 °C. Low intensity image of the substrate is also shown to better indicate substrate utilization. Note that similar inhibitory effects of #33 on the Nedd8-SCF^β-TrCP-dependent β-catenin ubiquitination were observed in at least five independent experiments. C, reactions with CRL1/SCF^β-TrCP. Incubation is 20 min at 37 °C. This experiment was repeated once. The detailed procedure is described under “Experimental Procedure.” The substrate levels after reactions were quantified and shown.