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. 2015 Aug 4;10(8):e0134996. doi: 10.1371/journal.pone.0134996

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© 2015 Tzeng et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 3 — (A) Schematic representation of the double-stranded DNA used for in vitro binding and reporter gene assays. (B) The effect of spacer nucleotides in PPARα and RXRα binding to PPRE DNA. Biotin-labeled PPRE was incubated with recombinant PPARα and RXRα. (C) The effect of the spacer nucleotide within the PPRE on PPARα/RXRα heterodimer formation. GST-fused PPARα was incubated with RXRα and double-stranded DNA comprising PPREs possessing different spacer nucleotides. DNA comprising a single hexad element (AGGTCA-A-AAATCA) was used as a control (SH). Pull-down assays were performed with GSH beads. The Western blot analyses results with short- and long-time exposure are presented to show the reduced PPARα/RXRα heterodimerization in C spacer (Short Ex) and to show PPRE oligonucleotide-independent PPARα/RXRα heterodimerization (Long Ex), respectively. (D) The effect of spacer nucleotides on PPARα binding. The indicated biotin-labeled double-stranded DNA with A as a spacer was incubated with recombinant PPARα and unlabeled competitors. (E) The effect of the 3’ extended region of the PPARα binding hexad element in PPARα DNA binding. The indicated biotin-labeled double-stranded DNA was incubated with recombinant PPARα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). (D-E) All signals without competitors are identical among the 4 panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blot/membrane. (F-G) The effect of a spacer nucleotide in PPARα ligand- (F) and PPARα-overexpression- (G) induced transcriptional activation. Minimal promoters with PPRE harboring different spacers were stimulated by the ligand and PPARα (n = 10–12). The triangle indicates statistical significance compared to PPRE(A) stimulated with the same dosage of WY14643 or transfected with the same amount of PPARα expression vector. Solid triangle: significantly reduced; open triangle: significantly enhanced. PPRE(A) is identical to PPRE(←) in Fig 2.