Sp1-binding sites mutations abrogate the repression of RLIM promoter by p53.

<p>(<b>A</b>) Schematic representation of the RLIM promoter luciferase reporter construct NP500-Luc and various mutated constructs with the Sp1 binding sites mutations (NP500-M1 to NP500-M1234). The four Sp1 binding sites S1 to S4 (wild type denoted as □ and mutations denoted a ▪) are shown. The sequences of the Sp1 binding sites are shown in the lower panel with the mutated sites underlined. (<b>B–D</b>) Cotransfection was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062832#pone-0062832-g005" target="_blank">Figure. 5B</a> using NP500-Luc and various mutated constructs with indicated Sp1-binding sites mutations, respectively. Luciferase assays were performed as described above. (<b>E</b>) EMSA analysis of the binding of Sp1 to RLIM promoter with different Sp1 binding site mutations (M1–M4). EMSA was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062832#pone-0062832-g003" target="_blank">Figure. 3D</a>. Lane 1: rhSp1+Biotin-labeled probes containing wild type Sp1-1 binding site +50-fold excess cold unlabeled competitor DNA. Lane 2: rhSp1+Biotin-labeled probes containing wild type Sp1-1 binding site. Lane 3: biotin-labeled probes containing wild type Sp1-1 binding site only. Lanes 4, 6, 8: rhSp1+Biotin-labeled probes containing mutant Sp1 binding site. Lanes 5, 7, 9: Biotin-labeled probes containing mutant Sp1 binding site only.