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• W3017003640 abstract "Prolidase (PEPD) is a ubiquitously expressed cytosolic dipeptidase that has the unique ability to cleave proline or hydroxyproline on the C-terminus of imidodipeptides and imidotripeptides. The reaction is vital for collagen degradation. Since collagen is the most abundant protein in the body, our objective is to get a better understanding of the enzymes involved in its turnover is crucial. In silico analysis on prolidase promoter showed that putative transcription factor of interest is KLF6. To test the hypothesis that the expression of PEPD gene may be regulated by the binding of the transcription factor KLF6 to candidate binding sites we used chromatin immunoprecipitation (ChIP) assay. HEK293T cells were transfected with FLAG-hKLF6 plasmid and the cellular lysates were obtained to measure the KLF6 protein expression by immunoblotting. After that, purified DNA from the ChIP assays were used in end-point PCR with primers targeting candidate KLF6-binding sites in PEPD promoter. The primers used were expected to yield a 615 bp amplicon. PCR products were resolved on 1% agarose/TAE gel. The second round of PCR was performed in which endpoint PCR products from the first round of PCR were used as a template for nested PCR with primers designed to amplify 164 bp. Nevertheless, our results showed no detectable PEPD promoter-specific signals in the PCR analysis of the ChIP products. However, due to the inherent caveats in the experimental design, choice of reagents, and analysis method, it would be premature to conclude at this time point that PEPD expression is not regulated by KLF6. Accordingly, the design of future experiments will effectively address the limitations of this current work and will help reveal the identity of transcription factors regulating PEPD expression. Support or Funding Information Supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni-Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527)." @default.
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• W3017003640 date "2020-04-01" @default.
• W3017003640 modified "2023-10-17" @default.
• W3017003640 title "Studying KLF6 and PEPD ‐gene Promoter by Chromatin Immunoprecipitation Assay" @default.
• W3017003640 doi "https://doi.org/10.1096/fasebj.2020.34.s1.04984" @default.
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