Fig 2.

GRIP1 phosphorylation requires a physical interaction with agonist-bound GR. (A) RU486 abolishes GRIP1 phosphorylation. U2OS-rGR cells were treated with 100 nM dex, 100 nM RU486, or dex plus RU486 (0.1 to 1 μM) for 2 h. GRIP1 and STAT3 were detected as described for Fig. 1B. (B) GRIP1 dex-induced phosphorylation requires the GR GRIP1-binding surface. U2OS cells stably expressing WT or E773R rGR were treated with or without dex, and GRIP1 mobility was assessed by immunoblotting. (C) The GRIP1 NR box-3 is required for GRIP1 phosphorylation. U2OS-rGR cells transiently transfected with an empty vector (−) or pcDNA3-GRIP1 expressing the mouse WT or NR box-3 mutant (box3m) GRIP1 were treated with or without dex for 2 h, and GRIP1 mobility was assessed by immunoblotting. At the exposure time selected, only ectopically overexpressed GRIP1 is detectable.