Figure 4.

Estrogen-dependent interaction of CITED1 with ERα in vivo. (A) CITED1 interacts with the LBD/AF2 domain of ERα in an estrogen-dependent manner. COS-1 cells were transfected with the FLAG-tagged AF1 or LBD/AF2 domain of ERα together with HA-tagged CITED1 and cultured for 24 h in the absence of hormones. Cells were then stimulated with 100 nM 17β-estradiol (E2) or vehicle (ethanol) for 1 h, followed by anti-FLAG immunoprecipitation. Coprecipitated HA-CITED1 was detected by anti-HA Western blotting (top). Expression of the transfected proteins was evaluated by anti-FLAG (for AF1 or AF2 domains) or anti-HA (for CITED1) Western blotting (bottom). (B) Transfected CITED1 interacts with endogenous ERα in an estrogen-dependent manner. MCF-7 cells were transfected with HA-tagged CITED1 and cultured for 24 h in the absence of hormones. Cells were then stimulated with 100 nM 17β-estradiol (E2) or vehicle (ethanol) for 1 h, followed by anti-HA immunoprecipitation. Coprecipitated ERα was detected by Western blotting using an anti-ERα antibody (top). Expression of proteins was evaluated by anti-HA (for transfected CITED1) or anti-ERα (for endogenous ERα) Western blotting (bottom).