Association of wild-type and mutant C/EBPα with the IGnTC gene 5′ promoter region. Wild-type C/EBPα and C/EBPα mutants with respective substitutions of S21A, S21D, S234A, and S234D were expressed in K-562 cells cultured in noninducing conditions. The association of wild-type and mutant C/EBPα with the 5′ promoter region (−322 to +62 bp) of the IGnTC gene in K-562 cells was examined by the use of ChIP analysis. A total of 1 × 10⁷ K-562 cells expressing wild-type C/EBPα, mutants C/EBPα-S21A, C/EBPα-S21D, C/EBPα-S234A, and C/EBPα-S234D or mock pcDNA3.1− vector were used. The chromatin DNAs were immunoprecipitated (IP) with a 1:500 dilution of anti-C/EBPα antibody and the input DNA controls were used in PCR amplification for the IGnTC −322 to +62-bp region.¹⁸  The products were analyzed by the use of 2.0% agarose gel electrophoresis. The K-562 cells expressing mock pcDNA3.1− vector were used for the no-antibody control in the PCR amplification, and only a trace of PCR product was observed (not shown). The results were analyzed by the use of densitometry, and the quantification analysis showed that the ratio of the band intensities (from left to right), after calibration with individual input controls, was 1.00:2.95:2.99:1.55:3.03:3.15.