Fig. 9

KLF6a and KLF7a bind the tuba1a G/C-rich element and can transactivate the tuba1a promoter through the G/C-rich element. (A) EMSAs were performed using radiolabeled G/C-rich element and in vitro synthesized KLF6a (6), KLF7a (7), myc-tag (MT), myc-tagged KLF6a (MT6), or myc-tagged KLF7a (MT7) protein. If no protein was added the lane is marked with a (-) and if the in vitro transcription translation reaction mix, lacking plasmid, was added the lane is marked with a (+). Both KLF6a and KLF7a bind to the probe (bracket) and in the cases where they harbor a myc-tag the anti-myc antibody (myc-Ab) results in a supershift of the complex (black arrow). (B) MT6 and MT7 binding is specific and can be completed by Wt oligonucleotide but not by M4 oligonucleotide (Wt and M4 EMSAs were performed on separate gels under identical conditions; M4 competitor concentrations are equal to the highest Wt competitor concentrations). Free probe (F). (C) KLF6a or KLF7a over expression can transactivate a heterologous promoter through the isolated G/C-rich element (*p < 0.01, t-test). (D) KLF6a or KLF7a overexpression can transactivate the tuba1a (α1T) promoter and this transactivation is attenuated when the G/C-rich element is deleted (Del - 166/-146) or mutated (M4) (*p < 0.01, ANOVA with Bonferonni post-hoc test comparing tuba1a (α1T) promoter driven expression with KLF6a or KLF7a overexpression, white bars) (#p < 0.05, ANOVA with Bonferroni post-hoc test comparing KLF6a or KLF7a overexpression effect on G/C-rich element deletion or mutation tuba1a (α1T):luciferase reporters, comparing grey or black bar to white bar). (C and D) PC12 cells were co-transfected with tuba1a (α1T):luciferase reporter vectors along with pCS2:β-gal and either empty pCS2 vector (Control) or pCS2-KLF6a (KLF6a) or pCS2-KLF7a (KLF7a). Reporter plasmids are: α1T, - 1696 tuba1a:luciferase; Del - 166/-146, internal deletion of the G/C-rich element in the - 1696 tuba1a promoter; M4, point mutant in G/C-rich element of - 1696 tuba1a promoter; MBG, minimal β-globin promoter; 4xWT, MBG with 4 copies of the G/C-rich enhancer cloned upstream; 4xM4, MBG with 4 copies of the M4 mutant G/C-rich enhancer cloned upstream. All transfections are normalized to pCS2:β-gal expression and luciferase values are reported as relative light units (RLU) in hundreds. Values are means +/- SEM.