CHOP inhibits the C/EBPα- and C/EBPϵ-mediated induction of expression of the secondary granule gene, B9, in NIH3T3 cells. NIH3T3 cells were transfected with expression plasmids pCDNA3.1 (empty vector control), CHOP, C/EBPα, and C/EBPϵ alone or in combination (total 4 μg), as noted. Total RNA was isolated 48 hours after transfection. (A) RT-PCR was carried out using gene-specific primers for the murine myeloid secondary granule gene B9. PCR products were separated in 1% agarose gel, transferred to a nylon membrane, and hybridized with a ³²P-labeled internal oligonucleotide probe. RT-PCR for 18S was carried out as an internal control. (B) Real-time PCR was performed on the same cDNA samples using B9- and 18S-specific primer and probes. Results are expressed in arbitrary units as a ratio of B9 transcripts/18S transcripts. The entire set of experiments was repeated at least 3 times using RNA samples made independently from separate cultures. Each value represents the mean of 3 measurements of the sample from a representative experiment.