Fig. 6.

The Pit-1 Mutant, R271A, Binds Specifically to DNA and Functions as a Dominant Inhibitor of PRL Gene Expression

A, HeLa cells were transfected with the rPRL luc reporter and the indicated amounts of the expression plasmids encoding C/EBPα, Pit-1, the Pit-1^R271A mutant, or the indicated combination. Luciferase activity was determined after 24 h and was corrected for total cellular protein. The error is the SEM from three independent experiments, each done in triplicate and normalized to reporter alone. B, GFP-Pit1^R271A was bound specifically to the PRL promoter 1P DNA element, but formed only a monomeric complex. Probe specificity was demonstrated using 3- to 100-fold excess unlabeled oligonucleotide (wedge, lanes 2–4), and immunoclearing was observed with either a GFP-specific (lane 5) or Pit-1 specific (lane 6) antibody; NS, Nonspecific complex. C, HeLa cells were transfected with the rPRL luc reporter and the indicated amount of GFP-Pit-1 and GFP-Pit1^mut. Inset, Western blot demonstrating that the GFP-Pit1 and GFP-Pit-1^R271A were expressed at equivalent levels. Error is SEM from three independent experiments, each done in triplicate and normalized to reporter alone.