Figure 5.

Direct binding of CITED1 to the LBD/AF2 domain of ERα in vitro: GST pull-down assay. (A) Estrogen-dependent binding of CITED1 to ERα. Equal amounts of GST or GST-fusion proteins of ERα domains (AF1 or LBD/AF2) were immobilized on glutathione-conjugated Sepharose beads and incubated with polyhistidine epitope-tagged CITED1 (His-CITED1) in the presence or absence of 100 nM 17β-estradiol. After extensive washing of the beads, coprecipitated CITED1 was detected by anti-His Western blotting. (B) Loss of ERα-binding activity of a function-dead CITED1 mutant. Equal amounts of GST-fusion LBD/AF2 of ERα were immobilized on beads and incubated with His-CITED1 or His-CITED1(mut2), a CITED1 mutant that does not enhance ERα-mediated transactivation (see Fig. 3), in the presence or absence of 100 nM 17β-estradiol. After washing of the beads, coprecipitated CITED1 was detected by anti-His Western blotting.