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Figure 7 | Arthritis Research & Therapy

Figure 7

From: Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression

Figure 7

Hemagglutinin-tagged peroxisome proliferator-activated receptor-gamma (HA-PPARγ) chromatin immunoprecipitation (ChIP). SW-1353 cells were transiently transfected with a construct expressing HA-PPARγ and treated for 24 hours with 50 nM LG268, 50 nM rosiglitazone, or both, followed by no treatment or 1 ng/mL interleukin-1-beta (IL-1β) for 24 hours. Cells were crosslinked with formaldehyde, and nuclei were collected and sonicated to shear chromatin to an average length of 500 base pairs. The crosslinked sonicated chromatin was immunoprecipitated overnight with an antibody to the hemagglutinin tag and pulled down with protein A/G agarose beads. The immunoprecipitated DNA was treated with Chelex 100 beads followed by proteinase K and used in real-time polymerase chain reaction with primers flanking the direct repeat-1/activator protein-1 (DR-1/AP-1) site of matrix metalloproteinase-1 (MMP-1) or MMP-13 or with negative-control primers flanking a region of DNA -3 kb upstream from the DR-1/AP-1 in MMP-1 or -1 kb upstream for MMP-13. Data were normalized to nonspecific IgG-precipitated DNA and expressed as fold-change over untreated cells. Results are representative of at least three independent experiments. 268, LG100268; NoTx, no treatment; PPARγ, peroxisome proliferator-activated receptor-gamma; Rosi, rosiglitazone.

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