Figure 5

Mutation analysis of transcription factor binding sites. A series of luciferase reporter constructs harbouring site-directed mutations in the NF-κB, NF-1 and Sp1 binding sites of the P2X₁ proximal promoter region were assayed for promoter activity in PMA-treated MEG-01 cells (sequences of mutations are shown in Table 2). Shaded symbols represent mutated binding sites. Values are presented as means ± S.E of at least three independent experiments and are normalized against the wild type p-190/+149 construct for panels A to C and the wild type construct p-4407/+365 in panel D. A) Effects of mutations in the construct p-190/+149. B) Effect of mutation after removal of the potential NF-κB binding site. C) Effect of mutation after removal of the transcription start site that is utilized by smooth muscle cells [15]. Dotted vertical arrow (TSS) represents the transcription start site determined in this study. D) The Sp1a, Sp1b and NF-1 sites were mutated in the original full length 4.7 kb reporter construct p-4407/+365 to generate the triple mutated construct p-4407/+365(mut4). Promoter activities were determined in MEG-01 cells either treated (+) or untreated (-) with PMA.