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. Author manuscript; available in PMC: 2021 Sep 3.
Published in final edited form as: Mol Cell. 2020 Jul 14;79(5):812–823.e4. doi: 10.1016/j.molcel.2020.06.031

Figure 4. The AR NTD is essential for SRC-3 and p300 coactivator recruitment.

Figure 4.

(A) AR interacts with SRC-3 through its N-terminal domain (NTD) in vitro. Purified flag-tagged full-length AR, AR(1–669aa) or AR (600–920aa) was incubated with purified, recombinant SRC-3 in the presence of 1 μM R1881 followed by co-immunoprecipitation using an SRC-3-specific antibody. (B) Deletion of two N-terminal domains of SRC-3, but not RID LXXAA mutations, abolished AR-SRC-3 interaction in vitro. Flag-tagged SRC-3 wild-type (WT) or mutant was transiently transfected into 293T cells and the lysates were incubated with purified, recombinant AR protein in the absence or presence of 1μM R1881 followed by co-immunoprecipitation using an AR-specific antibody. Red asterisks represent mutation of the three LXXLL motifs present in SRC-3 RID into LXXAA. (C) The SRC-3 RID does not interact with AR in a GST pull-down assay. Glutathione-sepharose beads bound to different GST-SRC-3 fragments were incubated with purified ERα, AR or ARV7 protein in the absence or presence of corresponding ligand. Top 3 panels, Immunoblotting analysis of glutathione bead-bound receptor proteins. Bottom panel, Coomassie blue staining of the different tested GST-SRC-3 fragments. (D) AR interacts with p300 through its N-terminal domain in vitro. Purified flag-tagged full-length AR, AR (1–669aa) or AR (600–920aa) was incubated with purified, recombinant p300 in the presence of 1 μM R1881 followed by co-immunoprecipitation using a p300-specific antibody. (E) SRC-3 increases the interaction between AR and p300 when both are at low concentrations. A series of concentrations of purified AR, p300, ARE-containing biotinylated DNA were incubated with or without purified SRC-3 in the presence of 1μM R1881 followed by magnetic streptavidin beads pull-down. Quantitation of p300 pulled-down by ARE DNA is provided underneath the top panel. (F) The effect of different concentrations of purified SRC-3 on the interaction between p300 and AR at low concentrations. Purified p300 protein was incubated with 1μM R1881, AR, and ARE DNA in the absence or presence of increasing concentrations of SRC-3 protein followed by magnetic streptavidin bead pull-down. See also Supplemental Figure 4.