Fig. 4. GRIP1 overexpression potentiates GR-mediated activation and repression of AP-1 activity in an NR box 3-dependent manner. U2OS.G cells were transfected in 24-well plates with indicated amounts per well of pCDNA3-GRIP1 (wt, S2A, S3A or 4X). Total amount of transfected DNA was equalized with pCDNA3 empty vector. Transcriptional activation of an XG₄₆TL [(A), 40 ng/well] or repression of AP-1-luc [(B) and (C), 40 ng/well] reporter was measured in the absence or presence of 100 (A and C) or 1 (B) nM dex. Endogenous AP-1 activity in (B) and (C) was stimulated with 25 ng/ml PMA. Luciferase activity is normalized to the β-gal activity of a co-transfected β-actin-LacZ plasmid (50 ng/well) and expressed as RLU. The y-axis in (C) is broken to visualize the effect of GRIP1 on GR-mediated repression at saturating dex concentration. Shown is a representative of two (B) or three (A and C) independent experiments performed in duplicate.