Figure 7. GAPDH enhances hTAF_II68-TEC-mediated transcription.

(A) Schematic representation of the reporter and expression plasmids used in the present study. The p(B1a)⁸-Luc reporter plasmid contains eight copies of NBRE upstream of a basal promoter-luciferase gene construct. The eight copies of hTAF_II68-TEC recognition sites are indicated by solid bars, the TATA box is represented by an open box, and the luciferase gene is indicated by a solid box. The expression vectors driving the production of hTAF_II68-TEC, GAPDH or GAPDH (217–335) are also shown. The positions of the first and last amino acids are indicated below each construct. CMV, cytomegalovirus promoter. (B) Stimulation of hTAF_II68-TEC-mediated transactivation by GAPDH. Aliquots of hTAF_II68-TEC (bars 1–4) were co-transfected into HeLa cells with 2 μg of empty vector (open bars) or GAPDH expression plasmid (black bars). After 48 h the cells were harvested and luciferase assays performed. The experiments were repeated three times, and the averages of two independent experiments are presented and error bars are shown. (C) A GAPDH (217–335) mutant lacking the hTAF_II68-TEC-interacting domain does not stimulate hTAF_II68-TEC-mediated transcriptional activation. HeLa cells were transfected with 1 μg of p(B1a)⁸-Luc reporter plasmid, 5 μg of hTAF_II68-TEC, and 5 μg of GAPDH (217–335) or empty vector, and luciferase activity was assayed. Relative transcriptional activation values were calculated with hTAF_II68-TEC taken as 100%. The experiments were repeated three times, and the average of two independent experiments are presented with error bars.