Figure 2. Activation of the BRE Reporter Plasmid by Grg4.

A) Pax2 reporter plasmid (PRS4) or pPax2/Bmp7 double reporter plasmids (PRS4+BRE) were co-transfected with Pax2 or with Pax2 and flag-Grg4. EGFP levels were assayed by western blotting. Note that Grg4 alone activates the double reporter but suppresses Pax2 activation of the single reporter (PRS4). B) The BRE element is sufficient to mediate Grg4 dependent activation. Two copies of the BRE element were inserted upstream of the TK-EGFP plasmid and co-transfected with either Pax2 or flag-Grg4 and expression of EGFP analyzed by western blotting. Note that Grg4 activates EGFP but in the absence of the PRS4 element Pax2 does not activate. C) Luciferase assays using the BRE-Luc reporter and co-transfection with flag-Grg4 or the addition of recombinant Bmp7. Conditioned media (cm) from GFP or Grg4 transfected cells was also tested for activation of BRE-Luc. Note that Bmp7 activates BRE-Luc approximately 3–4 fold but Grg4 expression activates more than 15 fold. D) Copy number and orientation of the BRE in transfected HEK293 or renal epithelial TKPTS cells. Copy number affects base level EGFP reporter expression but does not significantly impact Grg4 mediated activation. E) Increasing amounts of transfected Grg4 show increased levels of EGFP activation using the BRE-EGFP reporter.