Co-immunoprecipitation of SCF subunits with proteasome components. (A) Extracts from Hela cells were immunoprecipitated with a polyclonal antibody that recognizes multiple proteasomal subunits (lane 1) or a nonrelevant polyclonal serum (lanes 2). Lane 3 contains whole cell lysate. Immunoprecipitates were analyzed by immunoblotting with antibodies to the indicated proteins. A non-specific cross-reactive band is indicated by the asterisk. (B) Extracts from Hela cells were immunoprecipitated with a monoclonal antibody to alpha 6 (lane 1), a monoclonal antibody to alpha 2 (lane 4), a monoclonal antibody to alpha 4 (lane 5), a nonrelevant supernatant (lane 2) or mouse IgG (lane 6). Lanes 3 and 7 contain whole cell lysate. Immunoprecipitates were analyzed by immunoblotting with antibodies to the indicated proteins.

Co-purification of SCF subunits with proteasomes. Lane 1 contains purified 26S proteasomes, lane 2 contains purified 19S proteasomes, lane 3 contains purified 20S proteasomes and lane 4 contains whole cell lysate from Hela cells. Purified proteasomes and protein extracts were analyzed by immunoblotting with the antibodies to the indicated proteins.

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.

The N-terminus of Cul1 binds alpha subunits of the 20S proteasome. 293T cells were transfected with empty vector (lanes 1 and 7) or FLAG-tagged versions of full-length Cul1 aa 1-776 (lanes 2 and 8), Cul1 aa 1-300 (lanes 3 and 9), Cul1 aa 1-452 (lanes 4 and 10), Cul1 aa 1-645 (lanes 5 and 11) or Cul1 aa 324-776 (lanes 6 and 12). Extracts were immunoprecipitated with a monoclonal antibody to the FLAG epitope (lanes 1–6). Lanes 7–12 contain whole cell lysates from transfected cells. Immunoprecipitates and whole cell lysates were analyzed by immunoblotting with antibodies to the indicated proteins.

Ubiquitylated Cul1 binds the S5a subunit of the 19S proteasome. (A) Pulldown of ³⁵S Cul1 using GST alone (lane 1), GST-S5a (lane 2), GST-αlpha 6 (lane 3) or GST-αlpha 7 (lane 4). (B) Pulldown of ³⁵S wild-type Cul1 (lanes 1 and 2) or ³⁵S Cul1(K720A) (lanes 3 and 4) using GST alone (lanes 1 and 3) or GST-S5a (lanes 2 and 4). Ten percent input of ³⁵S wild-type Cul1 and ³⁵S Cul1(K720A) are shown in lanes 5 and 6, respectively. (C) Pulldown of ³⁵S wild-type Cul1 in vitro translated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of methylated ubiquitin (MeUb) using GST alone (lanes 1 and 3) or GST-S5a (lanes 2 and 4). Ten percent input of ³⁵S Cul1 in vitro translated in the absence or presence of MeUb are shown in lanes 5 and 6, respectively.