Figure 3. Mammalian one- or two-hybrid assays.

(A) Schematic of pGal4-TATA-Luc reporter vector, which bear five Gal4 response elements (5xGal4) juxtaposed to the simple TATA box promoter driving the expression of the firefly luciferase cDNA as the reporter. (B) One-hybrid assay was used to assess the intrinsic activation function of the amino-terminus of ERα (amino acids 1–180) or ERβ (amino acids 1–148), which is genetically fused to the Gal4 DNA-binding domain (Gal4). Constructs (300 ng) were transfected into HepG2 cells together with the pGal4-TATA-Luc reporter vector (500 ng). (B) Two-hybrid assay was used to assess the functional interaction between the amino- and carboxyl-termini of ERs. The carboxyl-terminal E/F domain of ERα (amino acids 301–595) or ERβ (amino acids 287–530) was genetically fused to Gal4 DBD, while the amino-terminal A/B region of ERα or ERβ was conjugated to the activation domain (AD) of viral protein 16 (VP16). The proteins were expressed in COS-1 cells together with pGal4-TATA-Luc reporter vector in the presence of 10⁻⁸ M E2 for 48h. The mean ± SEM depicts three independent experiments performed in duplicate. Panel C is a modified figure from Yi et al. [10] and used with permission from The Endocrine Society, Copyright 2005.