Figure 4.

DAX-1 Physically Interacts with GR

A, Coimmunoprecipitation in COS-1 cells. COS-1 cells were transfected with GR, DAX-1, or both GR and DAX-1. Cells were treated with vehicle (CON) or 100 nm dex for 1 h as indicated. Lysates were prepared and proteins immunoprecipitated (IP) with a mouse anti-GR antibody. Recovered proteins were resolved by SDS-PAGE and probed by Western blotting (W) using a rabbit anti-DAX-1 antibody. The membrane was then washed and reprobed with the mouse anti-GR antibody. Input levels of DAX-1 and GR are shown in lower two blots. B, Coimmunoprecipitation in A549 cells. A549 cells expressing endogenous GR and DAX-1 were treated with vehicle (CON) or 100 nm dex for 15 min. Lysates were prepared and proteins immunoprecipitated with a mouse anti-GR antibody or an equivalent amount of a mouse IgG1 antibody that does not recognize GR. Recovered proteins were analyzed as described above. C, GST pull-down assay. GST-GR deletion constructs were expressed in bacteria, and partially purified lysates were incubated in the presence or absence of dex with DAX-1, which was synthesized by in vitro transcription and translation reaction. The complexes were pulled down by glutathione-couple agarose beads, washed and resolved by Western blotting, and probed with antibodies against DAX-1. Blots shown are representative of two to four independent experiments. CON, control.