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Fig. 7 | Reproductive Biology and Endocrinology

Fig. 7

From: Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-kB, specificity protein (SP)1-like and GATA-4 binding sites

Fig. 7

A bp substitution at −1235 allows for NF-κB and SP1-like factor binding to the Meishan promoter. a Oligonucleotide probes (5’ to 3’) were synthesized containing sequence flanking a naturally occurring point mutation at −1235 of the Control/Index (C/I −1245/−1225) or −1232 of the Meishan (M −1243/−1223) promoter. Underlines within each DNA probe represent the nucleotide substitution between lines and putative NF-κB (TCCTCA; Blue) and SP1 (GGCGG; Red) elements identified by sequence analysis are highlighted. b EMSAs were performed by incubating radiolabeled oligonucleotides with nuclear extracts (5 μg) from αT3-1 cells and specificity of DNA-protein interactions was assessed by competition with 50-fold molar excess of homologous or heterologous unlabeled DNA (specific complexes indicated by arrows). Additionally, the DNA-protein complex was challenged by competition with 50-fold molar excess of unlabeled oligonucleotides containing consensus binding sequences for AP2, NF-κB and SP1. Electrophoresis of the gel was performed for an extended time, thus, free probe was run off the gel. c To determine the specific factors comprising the Meishan-specific complex, nuclear extracts were also incubated with antibodies directed against the p50, p52, and p65 subunits of NF-κB, SP1 (Upstate), SP1 (Santa Cruz Biotechnology), SP2, SP3, SP4 or an equal mass of rabbit IgG (supershift indicated by arrows)

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