Prdx6 could interact with C/EBPβ <i>in vivo</i>.

<p>Immunoprecipitation was performed in PK15 cells using anti-Prdx6 and anti-IgG antibodies. Total Protein was used as input. The protein from Normal rabbit IgG group was used as negative control. (A) Detection of C/EBPβ protein in immunoprecipitation. (B) Detection of CREB protein in immunoprecipitation.</p

The porcine Prdx6 gene promoter was analyzed by computational analyses together with luciferase reporter system.

<p>(A) Conserved sequences of transcription factor binding sites of <i>Prdx6</i> promoter. The aligned region is conserved among pig, human and bovine. (B) Six promoter constructs were transfected into C2C12, 3T3-L1 and PK15 cell lines, and assayed for luciferase activity. The pGL3-Control and pGL3-Basic were the positive and negative control respectively. Green, orange, blue, red and bright blue dots on the left panel represented the C<b>/</b>EBPβ, MyoD, CREB, Sp1 and HSF binding sites predicted on TFsearch or TESS websites, respectively. Data are expressed as means ± SD of three replicates. (C) Four further deletions (D5.1, D5.2, D7, and D8) were transfected into C2C12 cells and assayed for luciferase activity.</p

C/EBPβ and CREB could up-regulate the <i>Prdx6</i> expression.

<p>(A) The Prdx6 deletions were co-transfected with relative overexpressed vector or pcDNA3.1 empty vector. The promoter activity was measured and the results were expressed as means± SD of three replicates. (B) Schematic structure of site-direct mutants of <i>Prdx6</i> promoter linked to pGL3-Basic vector. (C) Wild-types and mutants of the <i>Prdx6</i> deletions were transfected into C2C12 cells, and luciferase activity was detected and the results were expressed as means± SD of three replicates. (D) The <i>Prdx6</i> mRNA expression level was detected by RT-PCR after overexpression of each transcription factor into PK15 cells, data were showed as means± SD of three replicates. (E-G) The transfection efficiency of C<b>/</b>EBPβ, CREB or HSF1 overexpression vector and their effect on target Prdx6 protein expression level were determined by western blot. (H-I) The interference efficiency of Knockdown of C<b>/</b>EBPβ or CREB and their effect on target Prdx6 protein expression level were determined by western blot. Quantification results of western blot represented by ratio of C<b>/</b>EBPβ, CREB, HSF1 or Prdx6 to β-actin protein expression level (Image J software).</p

C/EBPβ and CREB bind to <i>Prdx6</i> promoter region <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Binding of CREB with <i>Prdx6</i> promoter was detected by EMSA with C2C12 cell nuclear extracts. Probes of CREB binding sites were labeled with biotin and the mutated nucleotides were depicted in red color. Lane1 was a blank control that did not add nuclear extracts; Lane2 was experimental group. For Lane3 and 4, a 1-fold excess of unlabeled or mutant probe was added to the reaction complex. The Lane5 was added 2μg anti-CREB antibody in the complex. The DNA-protein complex and the super-shift bands were indicated by arrows (B) ChIP assay of CREB binding to the <i>Prdx6</i> promoter in PK15 cells. (C) The interaction of C<b>/</b>EBPβ with <i>Prdx6</i> promoter was detected by EMSA with the porcine IMF cell nuclear extracts. The Lane1-5 experimental groups are similar to figure 4A. (D) Binding of C<b>/</b>EBPβ on the <i>Prdx6</i> promoter was determined by ChIP assay.</p

Identification of <i>Prdx6</i> TSS using 5′ RACE.

<p>(A) The <i>Prdx6</i> mRNA expression profile was analyzed by RT-PCR. (B) 5′ RACE PCR product of 632bp was visualized in 1.5% agarose gel. (C) Eighteen sequencing results of <i>Prdx6</i> 5′ RACE clones are listed and the major TSS adenine was defined as +1.</p

Model for mechanisms of Prdx6 participates in adipogenesis, muscle differentiation or regeneration.

<p>C/EBPβ or CREB-mediated up-regulation of Prdx6 may participate in adipogenesis, muscle differentiation or regeneration through two pathways: (1), Prdx6 inhibits adipogenesis and muscle differentiation by scavenging ROS; (2), Prdx6 interacts with C/EBPβ to participate in adipogenesis and muscle regeneration.</p