Figure 4. Effect of Sp1 and Sp3 on the transcriptional activity of ncx1-Br after site-directed mutagenesis of the putative Sp1 sites and analysis of their binding on ncx1-Br sequence. a, The core consensus sequences of the five putative Sp1A–E sites are boxed, and the locations relative to the reported transcription start site are separately numbered. Mutated nucleotides of the particular sequences are underlined, and the replaced nucleotides are shown in italics below the wild-type sequences. The bent arrows indicate the reported transcription start site. b, c, Cortical neurons transiently overexpressing Sp1 or Sp3 were cotransfected with pGL3-ncx1 or pGL3-ncx1/Sp1Bmut, pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs. Twenty-four hours after transfection, neurons were lysed in 1× passive lysis buffer. Lysates were analyzed for luciferase activity. Luciferase activity was expressed as firefly-to-renilla ratio. Each column represents the mean ± SEM (n = 4). *p < 0.05 vs pGL3-ncx1. **p < 0.05 versus all. d, EMSA was performed incubating Cy5′-Sp1 consensus sequence probe without or with nuclear extracts from SH-SY5Y cells (CTL NEG and CTL, respectively, lanes 1 and 2). Competition experiments were performed with increasing molar excess (20- and 100-fold) of the unlabeled wild-type ncx1/Sp1B–E (ncx1/Sp1B, lanes 3 and 4; ncx1/Sp1C, lanes 7 and 8; ncx1/Sp1D, lanes 11 and 12; ncx1/Sp1E, lanes 15 and 16) and the mutant ncx1/Sp1B–E probes (ncx1/Sp1B, lanes 5 and 6; ncx1/Sp1C, lanes 9 and 10; ncx1/Sp1D, lanes 13 and 14; ncx1/Sp1E, lanes 17 and 18). Antibodies specific for Sp1 and Sp3 were added to the EMSA reaction as indicated (lanes 19 and 20). e–g, Nuclear extracts from SH-SY5Y cells were incubated with the Cy5′-tagged ncx1-Sp1C–E (CTL, lanes 1). Competition experiments were performed with increasing molar excess (100-fold) of the unlabeled wild-type ncx1/Sp1C–E (ncx1/Sp1C–E, lanes 2) and the mutant ncx1/Sp1C–E (ncx1/Sp1C–E, lanes 3). Nuclear extracts were incubated in the presence of anti-Sp1, anti-Sp3, and anti-Sp4 (lanes 4–6). h, i, Chromatin was prepared from SHSY-5Y cells transiently transfected with Sp1, Sp3, and pN3 (empty vector) expressing plasmids and with the pGL3-ncx1 or pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs. Chromatin from transfected cells was immunoprecipitated with Sp1 and Sp3 antibodies. Exogenous DNA containing either the ncx1-Br fragment (−340/151, pGL3-ncx1) or the mutated Sp1C–E sequence alone or in combination (pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs) was amplified using ncx1-Br forward II promoter-specific forward primer and a luciferase gene-specific reverse primer (LucNrev primer). The binding activity of Sp1 is graphically represented as the percentage of total input of chromatin DNA. *p < 0.05 versus cells cotransfected with pN3 and pGL3-ncx1. **p < 0.05 versus cells cotransfected with constructs overexpressing Sp1 or Sp3 and pGL3-ncx1. #p < 0.05 versus all. Each column represents the mean ± SEM (n = 3).