Fig 6. PNRC isoforms differ in their activity to stimulate nuclear receptor mediated transcription by ERα or SF1.

Cos-7 cells were maintained in phenol-red-free DMEM with 10 % charcoal stripped FBS (A and C), or in the regular DMEM with 10 % FBS (B). Cells were transiently co-transfected with 0.125µg reporter pGL3(ERE)₃-SV40 or pGL3(SF1)₃-SV40, 25ng pCI-hERα or 0.2µg pSG5-SF1, and 1µg pCI-PNRC isoform or pCI. The total amount of transfected DNA was kept constant by adding the corresponding empty vectors. The transfected cells were added with DMSO or the ligand. Twenty-four hours after transfection, the cells were harvested and assayed for the protein concentrations and their luciferase activities using the Luciferase Reporter Assay System. The activity of PNRC isoforms to stimulate ERα (A) or SF1 (B) meditated transcription was analyzed by measuring relative luciferase activity of 10 µg cell extracts. The data are expressed as Mean ± SD of triplicate transfections. (C), Comparison of the ability of PNRC isoforms to potentiate ERα mediated transcription in the presence of the ligand at different concentration. Cos-7 cells were transiently cotransfected with 0.2 µg reporter plasmid pGL3(ERE)₃-SV40, 10ng pCI-hERα, and different amount of PNRC isoform expression vectors (0.05µg, 0.2µg and 0.5µg). The transfection and the luciferase assays were performed as described for (A) and (B).