ERF: an ets domain name protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic activation. and repurified to exclude DNA contamination. One microgram of RNA was used as template for oligo-dTCprimed RT using Superscript reverse transcriptase enzyme (Life Technologies). Five percent (vol/vol) of the purified cDNA (corresponding to 50 ng of RNA) was used as template for quantitative RT-PCR. Primers used to amplify a 322-bp fragment of rat utrophin (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AJ002967″,”term_id”:”2960012″AJ002967, position 9659C9981) were RUTROF (5-CAGTATGTGGCCAGAGCACTATGA-3) and RUTROR (5-GCAGATTTCTTTGCTCTTCCTCC-3). As an internal control for efficiency of RT and quantification, we simultaneously amplified a 194-bp fragment of rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH; accession “type”:”entrez-nucleotide”,”attrs”:”text”:”M17701″,”term_id”:”204248″M17701, position 335C529) using the primers RGAPDHF (5-CCATGGAGAAGGCTGGGG-3) and RGAPDHR (5-CAAAGTTGTCATGGATGACC-3). PCR was performed using a 2-min denaturation at 94C followed by 20 or 25 cycles (for GAPDH and utrophin, respectively) of 94C for 30 s, 60C for 30 s, and 72C for 30 s, followed by 72C for Arhalofenate 7 min, conditions that had been optimized for exponential phase amplification of both transcripts. Reactions were also performed in parallel, adding 1 l of [32P]dCTP/100 l of reaction mixture, for measuring radioactive incorporation. Products were resolved on 2% agarose gels and photographed using ethidium bromide. Photographs were digitized using an Agfa (Mortsel, Belgium) Arcus II scanner Arhalofenate at 1600 dots per inch, and bands were quantified using ImageQuant 1.1 software (Molecular Dynamics) for the Macintosh Arhalofenate OS 7.5.3 (Apple Computer, Cupertino, CA). Radioactive PCR products were resolved on 5% acrylamide gels, dried and the radioactivity incorporated in bands quantified using a Storm PhosphorImager and ImageQuant 1.1 software. Comparable results were obtained in both cases. DNA Affinity Purification The utrophin promoter UtroNBox probe explained above was ligated using T4 DNA ligase to streptavidin magnetic particles that have previously been coupled to a 16-mer oligonucleotide and utilized for DNA affinity purification as suggested by the manufacturer (Boehringer Mannheim, Mannheim, Germany). Typically, 50 g of L6 myotube nuclear extract were incubated with UtroNBox coupled magnetic particles and eluted in 25 l of high-salt buffer [20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM (NH4)2 SO4, 1 mM DTT, 0.2% Tween 20 (wt/vol), and 2 M KCl]. The DNA-binding proteins were dialyzed to reduce the salt concentration using a 3500 molecular excess weight cutoff (Pierce) membrane before analysis. Statistical Analysis All data were subjected to Students test for Rabbit polyclonal to ADORA3 calculation of statistical significance. Where appropriate they were also subjected to an additional parametric test (ANOVA) as well as nonparametric (Wilcoxons rank sum) assessments for statistical significance. Statistical analysis was performed using Statview 5.0 (SAS Institute, Cary, NC). All data are graphically represented with controls normalized to 100 and increases (or decreases) shown as a percentage of control levels. Error bars are specified in physique legends as SEM or Arhalofenate SD. RESULTS Heregulin Activates Utrophin Expression To address whether heregulin regulates utrophin gene expression, we treated rat L6 myotubes with heregulin and processed Arhalofenate the cultures for quantitative RT-PCR. As shown in Figure ?Physique1,1, heregulin treatment increased the mRNA level of utrophin to 195% compared with control cultures. Although this technique is usually both sensitive and specific, it cannot distinguish whether the observed increase of utrophin mRNA is due to increased utrophin gene transcription or changes in mRNA stability. Open in a separate window Physique 1 Heregulin increases utrophin mRNA in skeletal muscle mass cultures. Differentiated L6 rat myotubes were incubated with 1 nM heregulin in PBS for 30 min along with controls. RNA was extracted, and quantitative RT-PCR performed. (A) Representative experiment showing the 322-bp utrophin fragment and the 194-bp GAPDH control fragment obtained by RT-PCR. (B) Results of radioactive quantification of four individual experiments taken together. The stippled bars represent utrophin mRNA levels in untreated cells, and cross-hatched bars represent the levels in heregulin (HRG)-treated cultures. Heregulin treatment increases the endogenous utrophin message in muscle mass cell cultures to 195% of control levels. Error bars show SEM; n = 4). Asterisks denote the results were statistically highly significant at p 0.001. To verify the increase.