The pRBM704V protein is defective for LXCXE-dependent interactions. (A) The ability of wild type or mutant forms of GST-RB to interact with TAg or E2F3/DP1 was determined by pull-down experiments. The input level of GST-RB proteins was determined by coomassie staining of SDS-PAGE gels, and the quantity of TAg or HA-E2F3/HA-DP1 that was precipitated with GST-RB was detected by western blotting. (B) Interactions were also assessed by co-immunoprecipitation of pRB with TAg or HA-E2F3/HA-DP1 proteins. In each case, the quantity of pRB present in precipitated complexes was determined by western blotting. UT indicates extracts that were generated from untransfected cells. (C) RB1 deficient cells transfected with wild type or mutant pRB were treated with cycloheximide, harvested at the indicated time points, and the quantity of pRB present was determined by western blotting.