Pdc Case Study

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METHODS
Cells — Normal Human Bronchial Epithelial (NHBE) cells were obtained from Lonza (Walkersville, MD) and were grown and maintained in bronchial epithelial basal medium (BEGM, Lonza) supplemented with growth factors and hormones as described in the manufacturer's instructions, at 37°C in a humidified atmosphere of 5% CO2-95% air. Cells were treated with 15d-PGJ2 or Vehicle (Veh) in basal medium for 24 h, nuclear extracts were prepared and protein concentrations were determined using the BCA Protein Assay kit (Pierce, Rockford, IL).

PPARδ Reporter Assay — To determine the activation of PPARδ by 15d-PGJ2 a PPARδ specific luciferase reporter assay was performed using PPARδ Reporter Assay System (IB00121; Indigo Biosciences, PA)
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The infrared signal was detected using an Odyssey Infrared Imager (LI-COR, Lincoln, NE).

Chromatin immunoprecipitation (ChIP) assay — The ChIP assay was performed using the SimpleChIP Enzymatic Chromatin Immunoprecipitation kit with magnetic beads (Cell Signaling Technology) as described previously (1). An equal amount of chromatin was incubated overnight at 4°C with antibodies against PPARδ, or against IgG as control (Santa Cruz Biotechnology). Then protein G magnetic beads were added and the chromatin was incubated for 2 h, at 4°C with rotation. An aliquot of chromatin that was not incubated with any antibody was used as the input control sample. Antibody-bound protein-DNA complexes were eluted and subjected to real-time PCR (1) with specific primers that amplify the PPRE and β-Actin in the promoter.

Western blotting — Western blotting was performed as described previously (2). Primary antibody against PPARδ was from Cayman Chemical. The secondary antibody IRDye-800CW (925-32211) and IRDye streptavidin (925-68079), IRDye Maleimide (929-80020) were from LI-COR. The infrared signal was detected using an Odyssey Infrared Imager.

In Vitro Electrophilic Addition Reaction — The ability of 15d-PGJ2 to form covalent electrophile-protein adducts was investigated in vitro by performing an in-vitro electrophilic addition reaction. NHBE cell lysates, recombinant PPARδ, and PPARδ ligand binding domain (PPARδ-LBD) proteins were incubated with indicated concentrations of
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