FIG 3.

Activation of Cybb is associated with direct binding of PU.1. (A) Induction of PU.1 expression in iBN cells increases Cybb mRNA transcript levels. Cybb mRNA transcript levels were determined using RT-qPCR of RNA prepared 72 h after induction with 1,000 ng/ml Dox. (B) PU.1 association with the Cybb promoter is inducible. Shown are ChIP-seq tracks without (−) and with (+) Dox at 72 h after PU.1 induction. The Cybb gene structure is shown below the bottom panel; boxes represent exons. The dotted box indicates an increased site of PU.1 association. The dot underneath the bottom panel indicates the location of the primer pair used for ChIP-qPCR analysis. (C) Quantification of PU.1 association with the Cybb promoter. Anti-PU.1 ChIP was performed 72 h after Dox induction. The left panel shows percent input determined by qPCR using primers recognizing the Cybb promoter. The right panel shows fold enrichment comparing percent input from anti-PU.1 to that from IgG ChIP for three biological replicates. (D) Quantification of PU.1 association with Cybb promoter using anti-FLAG ChIP performed 72 h after Dox induction. The left panel shows percent input determined by qPCR using primers recognizing the Cybb promoter. The right panel shows fold enrichment comparing percent input from anti-FLAG to that of IgG ChIP for three biological replicates. **, P < 0.005; ***, P < 0.001.