Chromatin immunoprecipitation

The analysis was performed using the ChIP Assay Kit (Upstate, Lake Placid, NY, USA). Briefly, 293t cells were sonicated until DNA fragments averaged between 200 and 1000 bp. Chromatin was subsequently immunoprecipitated with antibodies (2 μg) against IDO, and an equal amount of IgG was used as a negative control for nonspecific immunoprecipitation. Next, ChIP DNA fragments were purified, reverse-transcribed, and used as templates for PCR. The PCR products were analyzed using agarose gel electrophoresis. Antibodies used were anti-IDO antibody (Abcam) and normal rabbit IgG (Santa Cruz). Primers specific to the IDO region were as follows: F, TCTCGGGCTCAAGCAATTC; R, TTCCGTTTATCCAGTCATCTC.