Fig. 5
ERβ interacts directly with TDG. a Interaction of ERβ and TDG in GST-pulldown assays. GST-tagged ERβ or GST alone was immobilised on Glutathione Sepharose and incubated with recombinant TDG. The left panel shows representative western blots of the eluates using an anti-GST and an anti-TDG antibody. Quantification of four independent experiments is shown the right panel. Asterisk indicates significantly (p < 0.05) increased intensity of the band corresponding to TDG in the presence of ERβ. b Interaction of ERβ and TDG on far-western blots. GST-tagged ERβ (lanes 1 + 2), GST (lane 3), and GST-tagged SUMO-1 as a positive control (lane 4) were immobilised on a membrane and probed with recombinant TDG. Proteins were detected using antibody against GST (left panel) or TDG (middle panel). The right panel shows a membrane not probed with recombinant TDG. Asterisk marks unspecific bands. c Interaction between ERβ and TDG in yeast two-hybrid assays. ERβ fused to the AD and TDG fused to the BD of GAL4 were expressed in the yeast strain AH109. Serial dilutions of cells were spotted on control (SC-LEU-TRP, left panel) and selective medium (SC-LEU-TRP-HIS-ADE, right panel) to monitor activity of the reporter genes ADE2 and HIS3. As a positive control, murine p53 fused to GAL4 BD was used in combination with SV40 large T-antigen fused to GAL4 AD. The Gal4 BD and/or GAL4 AD alone served as negative controls (—). d Domain mapping for ERβ using yeast two-hybrid assays. Activity was tested in the yeast strain Y187 using lacZ as a reporter gene. Activity of the lacZ-encoded β-galactosidase leads to cleavage of X-gal and concomitant accumulation of a blue product (5,5′-dibromo-4,4′-dichloro-indigo). In addition to constructs as in B, individual ERβ domains (AB, CDEF, DEF) were fused to the GAL4 AD and used for transformation of Y187 cells. 106 cells were dropped onto SC plates lacking leucine and tryptophan. After 24 h of growth, cells were lysed and incubated with X-Gal for up to 17 h to monitor appearance of blue colour