Fig. 6. GRIP1 increases TRβ-mediated transcriptional activation but not repression of AP-1 activity. (A) TRβ-1 ectopically expressed in U2OS.GT cells is competent for activation and repression. U2OS.GT cells were transfected in 24-well plates with 40 ng/well of TRβ-responsive reporter (DR4 or 2× TRE, left and middle panel), or AP-1 reporters (AP-1-luc, Δ-73col1-luc and colA-luc, right panel), and 50 ng/well of the β-actin-LacZ plasmid. Luciferase activity was measured in the presence and absence of 100 nM triac, normalized to β-gal activity and expressed as RLU; endogenous AP-1 activity was stimulated with 25 ng/ml PMA. (B) Transiently overexpressed GRIP1 coactivates TRβ in an NR box 2-dependent manner. U2OS.GT cells were transfected with amounts indicated per well of pCDNA3-GRIP1 (wt, S2A, S3A and 4X), pCDNA3 to equalize total amount of DNA, 2× TRE reporter and β-actin-LacZ. TRβ-dependent activation of 2× TRE reporter was assayed in the presence or absence of 100 nM triac, normalized to β-gal activity and expressed as RLU. (C) GRIP1 does not potentiate TRβ-dependent repression of AP-1 activity. U2OS.GT cells were transfected with amounts indicated per well of pCDNA3-GRIP1 (or pCDNA3 vector), indicated AP-1 reporters and β-actin-LacZ. AP-1 activity in 25 ng/ml PMA was assayed in the absence and presence of 100 nM triac; luciferase activity of AP-1-luc and colA-luc reporters was multiplied by 10 for uniformity with the activity of Δ-73col1-luc reporter.