Fig. 6. RCE1 interacts with the SCF and the RUB activating enzyme. (A) [³²P]RBX1 (20 µl) was added to glutathione beads loaded with 3 µg of either GST or GST–RCE1. After washing, proteins retained on the beads were analyzed by SDS–PAGE. (B) Protein extracts were prepared from 35S::Myc-RCE1 seedlings. Proteins were recovered after pull-down with GST–RBX1 or immunoprecipitation with α-CUL1 antiserum and analyzed by immunoblot using α-Myc antiserum. (C) GST–RCE1 pulldowns were performed using protein extracts prepared from GVG::TIR1-Myc or 35S::LRF1-Myc seedlings. Recovered proteins were analyzed by immunoblot using α-Myc antiserum. (D) Protein extracts from 35S::Myc-RCE1 seedlings were immunoprecipitated with α-AXR1 antiserum and immunoblotted with α-Myc antibody. Lanes 1 and 3, Columbia extract; lanes 2, 4 and 5, 35S::Myc-RCE1 extract.