XAP2 interacts with ERα but has no effect on the intracellular level of ERα.

(A) HC11 cells where transiently transfected with XAP2 siRNA (msiXAP2) (lanes 2,4) or a scrambled siRNA (Scr) (lanes 1,3). 48 h after transfection, cells were treated with DMSO (−E₂) or 10 nM E₂ (+E₂) for 1 h before harvest. Whole cell extracts were prepared and Western blot experiments were performed with indicated antibodies; β-actin was used as a loading control. (B) The ERα protein levels shown in (A) were quantified by measuring the density of specific bands and normalizing to β-actin progein levels. The ERα/β-actin ratio in Scr (−E₂) cells was arbitrarily set to 1 and data were expressed as means ± SE of three independent experiments. (C) HC11 cells were treated for 1 h with DMSO (−) or E₂. Whole cell extract (WCE) was prepared and immunoprecipitation (IP) experiments were performed using a XAP2 antibody (lanes 3–4). The presence of ERα protein was monitored by Western blot analysis. WCE (lane 2) and an IgG antibody (lane 1) show the positive and negative controls, respectively. Data shown here is representative of three independent experiments. (D) Radioactively labeled proteins XAP2 and ERα synthesized by in vitro translation were mixed in equal amounts. After incubation (for details, see Materials and Methods), XAP2 antibodies or IgG antibodies were added to each protein mixture. Precipitated complexes were analyzed by SDS-PAGE. Data shown here is representative of three independent experiments.