FIGURE 4. GABP regulates Il7r transcription in pro-B cells.

A, an Il7r reporter construct is activated by ectopic expression of PU.1 or GABP subunits. 38B9 pro-B cells were transiently transfected with equimolar amounts of DNA containing a pGL3-Basic vector containing the −925 Il7r promoter, as well as pCDNA3 expression vectors encoding PU.1, GABPα, GABPβ1, or both GABPα and GABPβ1. Empty pCDNA3 vector was used as a control and to equalize amounts of DNA. Transfections were normalized by transfecting equimolar amounts of a Renilla luciferase plasmid. Results are expressed as-fold activation above baseline reporter activity. Error bars represent standard deviation of the mean. B, RNA interference of GABPα transcription in pro-B cells. 220–8 pro-B cells were transiently transfected with empty expression vector (filled bars) or expression vector encoding short hairpin RNAs directed against GABPα (open bars). 60 h after sorting transfected cells, total RNA was prepared and real-time RT-PCR was performed to measure levels of mRNAs encoding either GABPα (left bars) or IL-7Rα (right bars). C, inducible RNA interference of GABPα transcription in pro-B cells. 38B9 cells infected with vectors encoding a reverse tetracycline-transactivator protein (rtTA, filled bars), or both rtTA and a tetracycline-regulatable vector expressing an anti-GABPα microRNA (open bars) were induced with doxycycline (Dox). After 72 h, total RNA was prepared and real time RT-PCR was performed to measure levels of mRNAs encoding either GABPα (left bars) or IL-7Rα (right bars). D, flow cytometric analysis of cells described in C.38B9 cells were analyzed for expression of cell-surface expression of CD43 or IL-7Rα.