Anti c jun

ChIP experiments were conducted according to the ChIP kit instructions (Upstate, Charlottesville, VA, USA). CAVO3 cells were fixed in 1% formaldehyde for 10 min for crosslinking reaction which was quenched with 125 nM glycin. The nuclei were pelleted by centrifugation at 3,000 rpm for 5 min at 4°C and sonicated to chromatin fragments between 100 and 1,000 bp. The sonicated lysate was centrifuged at 10,000 rpm for 5 min at 4°C. Supernatant (500 µl) was incubated with anti-c-Jun (1:50; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-c-Fos (1:50) antibody followed by an isolation procedure using protein A/G magnetic beads (both from Santa Cruz Biotechnology, Inc.). A normal rabbit IgG was used as a control. The crosslinking was reversed by incubation at 65°C for 10 h. Primers were designed according to the binding site of AP-1 in the FUT1 promoter and the sequence was as follows: F, 5′-CTAGCACTCAAGGTCCTGGTC-3′ and R, 5′-GCAAGATGAGGAAACTGAGGC-3′. The PCR conditions were as follows, 98°C for 5 min, 98°C for 30 sec, 60°C for 20 sec and 72°C for 5 min for 30 cycles. PCR products were resolved by electrophoresis on a 1% agarose gel and visualized after ethidium bromide staining.