Figure 3.

Transcriptional activities and DNA binding of WT and mutant GCM2 proteins with a synthetic GCM responsive promoter (3X-gbs). (A) Promoter–reporter construct containing three consensus GCM response elements in tandem upstream of a luciferase reporter gene (3xgbs-lux) was transfected into HEK293 cells with vector, WT or mutants (R67C, R67H, R67S or R47L). After 48 h, cells were harvested and luciferase activity of extracts measured. Values are mean ± s.e.m. of six estimations. (B) R67C mutant loses ability to bind to DNA. Biotinylated oligonucleotide pull-down assays were conducted on extracts of HEK293 cells that had been transfected 48 h before harvesting with either empty vector alone or Flag-GCM2 WT or mutants. The oligonucleotides used represented the WT and mutant GCM consensus element.