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Figure 7 | BMC Molecular Biology

Figure 7

From: Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus

Figure 7

Mutational analysis of USF elements in the AβH-J-J P1 promoter. (A) EMSA were performed incubating P1/USFmer probe with 2 μg of nuclear extract from HeLa cells in the absence (lane 1) or in the presence of 100 fold molar excess of the indicated competing oligonucleotides (lanes 2–3). (B) USFmer probe was incubated with 2 μg of nuclear extract from HeLa cells in the absence (lane 4) or in the presence of 100 fold molar excess of the indicated competing oligonucleotides (lanes 5–7) (Table 1). Arrows and asterisks indicate the specific and aspecific complexes respectively. (C) The reporter construct -512 WT (H in Fig 3) was subjected to site directed mutagenesis in the sequence spanning Sp1 and USF binding sites. The constructs obtained were designated as -512 P1/Spmut and -512 P1/USFmut. In addition a double mutant containing both mutations was indicated as -512 P1/Sp+USFmut. HeLa cells transient transfection and luciferase assay were performed in two experiments conducted in triplicate and the data were normalized to Renilla luciferase activity and reported as ratios (means ± SD) to the wild type reporter construct -512 WT; *P < 0.05 and **P < 0.02.

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