Figure 3.

Rbx1/ROC1 enhances the E3 activity of VBC-CUL-2. Hi Five cells infected with recombinant baculoviruses encoding CUL-2, elongin B (Elo B), elongin C (Elo C) together with either pVHL (lane 2), or Rbx1 (lane 4) or both (Lane1). Lysates of infected cells or noninfected cells (lane 3) were immunoprecipitated with anti-Flag M2 beads. (A) Immunoprecipitates were subjected to in vitro ubiquitination assay as described in Fig. 1C. Samples were electrophoresed in 10% SDS/PAGE, followed by autoradiography. (B) Immunoprecipitates were electrophoresed in 4–20% gradient SDS/PAGE. After being transferred, the membrane was probed with anti-HA, anti-Flag M2, anti-HSV, or anti-T7 antibodies, to detect CUL-2, pVHL, elongin C (Elo C), or Rbx1, respectively.