Recruitment of CREB to IL-11 gene promoter was determined by ChIP assay as described previously (24, 43, 44). Briefly, normal splenocytes were treated with aspirin under serum-free conditions, and after 3 hours of stimulation, cells were fixed by adding formaldehyde (1% final concentration), and cross-linked adducts were resuspended and sonicated. ChIP was performed on the cell lysate by overnight incubation at 4°C with 2 μg of anti-CREB, anti-CBP, anti-RNA polymerase II, or anti-p300 antibodies followed by incubation with protein G agarose (Santa Cruz Biotechnology) for 2 hours. The beads were washed and incubated with elution buffer. To reverse the cross-linking and purify the DNA, precipitates were incubated in a 65°C incubator overnight and digested with proteinase K. DNA samples were then purified and precipitated, and precipitates were washed with 75% ethanol, air-dried, and resuspended in tris-EDTA (ethylenediaminetetraacetic acid) buffer. The following primers were used for amplification of chromatin fragments of mouse IL-11 gene: 5′-CCGGGCGGGCTTCCCTCTCCCTCC-3′ (sense) and 5′-GGCTAGGGCTCCCGGGGCAGGGGA-3′ (antisense).

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