FIG. 5.
The C terminus of CUL1 interacts with ROC1 in a region that includes the cullin/APC2 consensus motif. (A) Immunoprecipitation analysis of Flag-CUL1 (324 to 776). 293T cells were transfected with HA-ROC1, Flag-CUL1, or Flag-CUL1 (324 to 776), alone or in combination. 35S-labeled extracts (approximately 0.2 mg of protein) were immunoprecipitated with αHA (lanes 1 to 5) or αFlag antibodies (lanes 6 to 10). Immunoprecipitates were separated by SDS–12.5% PAGE followed by autoradiography. (B) Immunoprecipitation and immunoblot analyses of Flag-CUL1 (324 to 776). Approximately 1 mg of extract protein was immunoprecipitated by M2 antibody cross-linked beads, and the resulting precipitates were examined by immunoblot analysis for the presence of Flag-tagged CUL1 derivatives, Skp1, HA-ROC1 and endogenous ROC1. (C) The ROC1 binding domain coincides with the cullin/APC2 homology region. 293T cells were transfected with CMV-based vectors expressing HA-ROC1, Flag-CUL1, Flag-CUL1 (1 to 452), Flag-CUL1 (1 to 544), or Flag-CUL1 (1 to 645), alone or in combination. 35S-labeled extracts (approximately 0.6 mg of protein) were immunoprecipitated with αFlag antibodies, and the immunoprecipitates were separated by SDS–12.5% PAGE. The portion of the gel containing both Skp1 and HA-ROC1 was exposed for four times as long as that containing CUL1.