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. 2000 Jun 27;97(14):7738–7743. doi: 10.1073/pnas.130198397

Figure 1.

Figure 1

p120E4F associates with pRB. (A) Recombinant p120E4F associates with cellular pRB. GST pull-down assays were performed with nuclear extracts prepared from confluent cultures of either RB+/+ (lanes 1, 3, and 5) or RB−/− NIH 3T3 mouse fibroblasts (lanes 2, 4, and 6). Extracts were incubated with either Sepharose-bound GST–p120E4F (lanes 3 and 4) or GST–ELK (negative control) (lanes 5 and 6). Bound proteins were released and probed for the presence of pRB by immunoblotting. One-third of the input extract was loaded in lane 1 and 2. (B) Recombinant p120E4F associates with the hypophosphorylated form of pRB present in arrested cells and does not associate at all with cellular p107 and p130. GST pull-down assays were performed as above by using nuclear extracts from either quiescent (lanes 1 and 3) or exponentionally growing (lanes 2 and 4) NIH 3T3 fibroblasts. Bound proteins (lanes 3 and 4) were probed for the presence of pRB (Top), p107 (Middle), or p130 (Bottom) by immunoblotting. One-third of the input extract was loaded in lane 1 and 2. (C) p120E4F is easily detectable in U2OS cells. Nuclear extract from U2OS cells is probed by immunoblotting by using an affinity-purified rabbit polyclonal antibody (E4F-88) (lane 3). Specificity of this Ab is tested on nuclear extracts prepared from a Chinese hamster cell line (CCL39-tet/E4F) expressing a tetracycline inducible p120E4F gene (lanes: 1, + tetracycline; 2, − tetracycline). (D and E) Coimmunoprecipitation of cellular p120E4F and pRB proteins from U2OS cells extracts. (D) p120E4F is immunoprecipitated from confluent U2OS nuclear extracts by using the E4F 88.2 Ab, and precipitate is probed for the presence of pRB by immunoblotting (lane 2). The same experiment is performed with the corresponding preimmune serum (lane 3). One-tenth of the input extract was loaded in lane 1. (E) pRB is immunoprecipitated from confluent U2OS nuclear extracts by using the anti-pRB mAb coupled to agarose beads and precipitate is probed for the presence of p120E4F by immunoblotting (lane 4). The same experiment is performed with control beads coupled to unrelated mAbs (lanes 2 and 3). One-thirtieth of the input extract was loaded in lane 1.