Structure characteristics of the bovine <i>PDHB</i> gene.

<p>a. Here we show the genomic, mRNA and protein components in detail. 5’- UTR:5’- untranslated region, 3’- UTR:3’- untranslated region, ORF: open reading frame, Transketpyr: transketolase, pyrimidine binding domain. b. 5’-RACE. Lane 1 and 2 are products of the first and second PCR, respectively. Lane M represents the marker of DL2000. c. 5’-regulatory region sequence of bovine <i>PDHB</i> gene. Arrows mark the transcription initiation sites. The cytosine residue is designated as +1. The transcription factor binding sites are boxed. The primers are underlined with the respective names below the line. The CpG island is indicated with red color.</p

Spatial expression analysis of bovine <i>PDHB</i> mRNA.

<p>We normalized the mRNA expression levels of <i>PDHB</i> to those of <i>GAPDH</i>. Error bars represent the standard deviation (SD) (n = 3).</p

ChIP assay of MYOG and C/EBPß binding to PDHB promoter in vivo.

<p>We analyzed immunoprecipitated products for MYOG (a) and C/EBPß (b) antibodies via RT-PCR. We analyzed immunoprecipitated products for MYOG (c) and C/EBPß (d) antibodies via ChIP-QPCR. We used total chromatin from muscle (a and c) and fat (b and d) as the input. We used normal mouse IgG as the negative control antibodies. **, P<0.01. Error bars represent the SD (n = 3).</p

Functional analysis of the mutated MYOG and C/EBPß sites.

<p>We transferred the mutated sites MYOG and C/EBPß into C2C12 myotubes (a) and 3T3-L1 cells (b). **, P<0.01. Error bars represent the SD (n = 3).</p

Promoter activity analysis of the bovine <i>PDHB</i> gene.

<p>a. We transferred six serial deletion constructs in pGL3-basic into C2C12 cells. After 5 h we replaced the transfection mixture with DMEM with 5% FBS (myoblasts) or 2% HS (myotubes). b. We transferred the same constructs into 3T3-L1 cells. We normalized relative luciferase activities to Renilla luciferase activity. The transcription factor binding sites of MYOG and C/EBPß are indicated with closed circles and ellipses, respectively. *, P<0.05. Error bars represent the SD (n = 3).</p

Primers utilized in this study.

<p>Primers utilized in this study.</p

Multi-alignments sequence analysis of the core functional promoter of bovine <i>PDHB</i> in relation toother mammals.

<p>The transcription factor binding sites are marked with boxes. The nucleotide sequence is numbered in 5'-regulatory sequence of the bovine <i>PDHB</i> gene (GenBank No. KJ649747).</p

EMSA involving 5’-biotin labeled MYOG and C/EBPß probes.

<p>a. 5’-biotin labeled MYOG probes and nuclear extracts of C2C12 myotubes. Lane 1: MYOG probes; lane 2: MYOG probes with nuclear extracts of C2C12 myotubes; lane 3: MYOG probes and nuclear extracts with a 125-fold unlabeled MYOG oligonucleotides; lane 4: MYOG probes and nuclear extracts with a 125-fold unlabeled mMYOG oligonucleotides. lane 5: MYOG probes and nuclear extracts with myogenin antibodies. b. 5’-biotin labeled C/EBPß probes and nuclear extracts of 3T3-L1 cells. Lane 1: C/EBPß probes; lane 2: C/EBPß probes with nuclear extracts of 3T3-L1 cells; lane 3: C/EBPß probes and nuclear extracts with 125-fold unlabeled C/EBPß oligonucleotides; lane 4: C/EBPß probes and nuclear extracts with 125-fold unlabeled mC/EBPß oligonucleotides. lane 5: C/EBPß probes and nuclear extracts with C/EBPß antibodies.</p

Absorbance of anti-DBP antibody bound to hapten-coated polystyrene wells.

<p>Absorbance at 492 nm under different concentrations of EDC and conjugation temperatures. Each data represents the mean of ±SD of three replicates (three plates on the same day).</p

Titers and sensitivity of antisera of immunized mice.

<p><i>^a</i>The coating concentration was 1 µg/mL.</p><p><i>^b</i>The last dilution of serum that gave an absorbance at 492 nm of >1.0 was judged as the titer.</p><p><i>^c</i>IC₅₀ represents the mean of 3 wells replicates.</p