FIG. 4.

Defining the domain of ERα required for interaction with and inhibition of the APPct complex. (A) Full-length ERα (lanes 1), ERα with a truncated N-terminal A/B region (ER179C) (lanes 2), ERα with a truncated C-terminal LBD (ER282G) (lanes 3), and an ERα mutant with inactive AF-2 (ER-TAF1) (lanes 4) were produced together with Fe65 by in vitro transcription/translation reactions in the presence of E2. GST pull-down assays were performed. Precipitated proteins were detected by immunoblotting analyses with anti-ERα (C-terminal, top panel), anti-ERα (N-terminal, middle panel), and anti-Fe65 (lower panel) antibodies. Coomassie blue (C.B.) staining data were included to show that similar amounts of GST and GST-APPct were used for the pull-down assays. (B) HeLa cells were transfected and treated as described in the legend to Fig. 1A, except that 0.3 μg of PRST7-hERα or a deletion mutant was used for transfections. Luciferase activity was determined and normalized with cognate β-galactosidase activity.