Fig 1: Halo-ChIP analysis of the PAI-1 promoter.Halo-tagged fusion constructs of PXR, RXRa, and FoxO3a were made as described in Materials and Methods and transfected into HeLa cells. Atorvastatin (PXR/RXRa) or insulin (FoxO3a) were added for the times indicated and chromatin was cross-linked using 1% formaldehyde for 10 m. The chromatin was then sheared and the HaloTag containing protein-DNA complexes were isolated using HaloLink resin (Promega). The DNA was then de cross linked by heating at 60°C overnight. The isolated DNA was then passed through a column (Fermentas) to remove any contaminating proteins and qPCR was performed as described in Materials and Methods. A. Schematic of the PAI-1 promoter with hypothetical binding sites and primers used in pPCR analysis (A, top) and the sequence of the PAI-1 promoter in this region (A, bottom). The green colored box is the putative nuclear receptor response element. The yellow box is the Fox binding element. The rose colored box is the TATA element. Arrows under the sequence show the direction and sequence of PCR primers. B. Results with primers amplifying the distal promoter. C. Results using primers to the proximal promoter.