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Figure 5 | BMC Developmental Biology

Figure 5

From: RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis

Figure 5

Functional characterization of Rfx2 MBS sites. (A). Binding competition for Rfx2 MBS by gel mobility shift assay. The MBS1 oligo was used as labeled probe for the bacterially expressed A-MYB DNA binding domain-maltose binding fusion protein. Lanes show increasing amounts of unlabeled competitor oligonucleotides in the range of 50 to 500 ng. (B). Stimulation of Rfx2 promoter fragments by co-transfection of A-myb expression vector. Transient transfections were carried out with or without the C-terminal truncated A-myb expression vector. The locations of the promoter fragments are indicated in Fig. 4C. Note that the scale for PFB and PFC is amplified 2-fold. (C). Ability of individual Rfx2 MBS multimers to promote expression of a luciferase reporter. Individual MBSs were ligated in a head to tail fashion to generate 5 copies in a minimal promoter luciferase vector. Results are of transient transfection plus or minus co-transfection with the A-myb d304 expression plasmid. Fold activation is indicated above the (+) columns. (D). ChIP assay shows in vivo Rfx2 promoter occupancy by A-MYB. Left: Amplification of Rfx2 promoter following chromatin capture by anti-A-MYB or IgG control. Right: Amplification of unrelated Tcrd locus following chromatin capture.

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