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Chromatin isolated from neonatal rat cardiomyocytes was immunoprecipitated with specific antibodies directed against anti-c-Jun/AP1 (Calbiochem) and anti-MEF2 (Ana Spec) using the EZ chromatin immunoprecipitation (Ch IP) assay kit (Upstate) and analysed by PCR using the following primer pairs: CNS13 Ch IP primers 5′-CCT TGG TTG TGA GTC TGT GTC T-3′ (forward) and 5′-AGT GGA GAG ACT GCT CTT TTC C-3′ (reverse) and control Ch IP primers 5′-AAT GGG ACT CCT CTT ACT CAT TTC T-3′ (forward) and 5′-AAA GTC CCT GAT GAC ACA CTA GTT C-3′ (reverse).Adeno-associated virus (AAV) vector plasmids contain an expression cassette consisting of a CMV promoter followed by the first intron of human growth hormone, target c DNA, woodchuck hepatitis virus post-transcriptional regulatory element (Gen Bank accession no.Transcriptional activities were obtained from three separate assays performed in quadruplicate.The myc-tagged mouse c-Jun/AP1 and MEF2C proteins were , 0.1% Nonidet P-40, 5% glycerol, 5 m M dithiothreitol, 1 m M EDTA, and 1 µg poly(d I-d C).The score for the matches were AP1: core match 1.000; matrix match 0.923. () A schematic diagram of control, inverted, and tandem CNS13 constructs.

We prepared CNS13 reporter constructs harbouring mutations within the MEF2 and AP1 sites (, a single mutation in either MEF2- or AP1-binding sequences significantly reduced transcriptional activity, whereas double mutations completely abolished CNS13-induced enhancement.

We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression.

The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13.

The probe (10 fmol) incubated with 1 µL of protein was analysed on 4% polyacrylamide gels in ×0.25 TBE buffer.

In competition experiments, 100-fold molar excess of double-strand oligonucleotides (AP1: 5′-ATT CTG AGT CAG AGA-3′ and MEF2: 5′-AGG TGG GTT AAA AAT AGA GCC CT-3′) were added.

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