Figure 7.

FoxP1/2/4 bind and activate the very low density lipoprotein receptor (VLDLR) promoter. DNA binding assays with FoxP1 (A) and FoxP4 (B). Nuclear extracts (1 μg) from HeK293 cells were incubated with the digoxigenin labeled probe (0.8 ng) representing the 27-bp of the VLDLR FoxP2 binding site. Shown in each case are protein lysate of HeK293 cells transiently transfected with empty vector and labeled probe (lane 1), shift in the presence of nuclear extract of FoxPs (lane 2), and complex formation in the presence of 200-fold molar excess of specific un-labeled probe (lane 3) and supershift in the presence of labeled probe, FoxPs protein extract and monoclonal V5 antibody (1 mg/ml; lane 4). In all cases arrows point to free oligo, non specific shift (n.s.), FoxP shift and supershift. (C) Luciferase assays were carried out in HeK293 cells to measure effects of a FoxP1/2/4 alone or in combinations on the VLDLR promoter. Significance levels from all combinations to the empty vector control are represented by asterisk, **p < 0.001–0.01; ***p < 0.001. One way analysis of variance (ANOVA); F = 26.09; DF = 7 and n = 8; followed by Tukey’s multiple comparison. Bars show mean of means ± SEM of four independent transfections, presented as luciferase/renilla ratio (RLU), corrected for transfection by pGL4.75 Renilla luciferase activity. 1x = 125 ng of overexpressing vector per well, 2x = 250 ng of overexpressing vector per well. The control transfection value was obtained with the empty expression vector (pcDNA3.1).