Figure 5

Cul1 ubiquitylation promotes binding to the proteasome, but not degradation. (A) 293T cells were transfected with His-tagged ubiquitin (lanes 1 and 4), HA-tagged Cul1 (lanes 2 and 5) or His-Ubiquitin and HA-Cul1 (lanes 3 and 6). Cell extracts were subjected to nickel agarose purification (lanes 1–6). Precipitates were analyzed by immunoblotting with an antibody against the HA tag (lanes 1–3) or an antibody to Cul1 (lanes 4–7). Lane 7 contains recombinant Cul1. The bracket on the left side of the images marks a ladder of bands >90,000 corresponding to ubiquitylated Cul1. (B) 293T cells were transfected with ubiquitin (UbWT) (lane 1), a Ub(K0) mutant (lane 2), HA-tagged Cul1 (lane 3), UbWT and HA-Cul1 (lane 4) or Ub(K0) and HA-Cul1 (lane 5). Cell extracts were immunoprecipitated with an anti-HA antibody. Precipitates were analyzed by immunoblotting with an antibody to Cul1 (lanes 4–7). The lower image represents a short exposure time and the upper image represents a long exposure time. The bracket on the left side of the images marks a ladder of bands >90,000 corresponding to ubiquitylated Cul1. (C) 293T cells were transfected with HA-Cul1 in combination with empty vector (lanes 1–7) or Ub(K0) mutant (lanes 8–14). Cells were incubated in the presence of cycloheximide (CHX) for the indicated times. Degradation of Cul1 was monitored by immunoblotting with an anti-HA antibody. The asterisk indicates the neddylated form of Cul1. (D) 293T cells were transfected with HA-tagged Cul1 plus wild-type ubiquitin (UbWT) (lanes 1–2 and 5) or Ub(K0) mutant (lanes 3–4 and 6). Lysates were subjected to a pulldown using GST alone (lanes 1 and 3) or GST-S5a (lanes 2 and 4). Lanes 5 and 6 contain whole cell lysate. Pulldowns and lysates were analyzed by immunoblotting with an antibody to Cul1. The bracket on the left side of the images marks a ladder of bands >90,000 corresponding to ubiquitylated Cul1.