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Transfection of Rat Intestinal Epithelial Cells with a Stable Mitogen‐Activated Protein Kinase Phosphatase (MKP)‐1 Mutant
Author(s) -
Talavera Maria Margarita,
Crowell Sara,
Nelin Leif Daniel,
Liu Yusen
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1023.9
Subject(s) - transfection , microbiology and biotechnology , biology , mapk/erk pathway , protein kinase a , mutant , expression vector , innate immune system , immune system , serine , phosphorylation , biochemistry , gene , recombinant dna , immunology
MAPK phosphatases (MKPs) are critical modulators of the innate immune response in both immune cells and intestinal epithelial cells (IECs). MKP's regulate the innate immune response via MAPK inactivation by dephosphorylation of specific Tyr‐Xaa‐Thr motif, where Xaa represents amino acid residues specific to a MAPK subfamilies. We have shown that peak MKP‐1 expression in LPS stimulated rIEC‐6 cells was 60 minutes, limiting the regulation of the inflammatory response to a short interval. The objective of this study was to create a unique transfected rIEC‐6 cell line that expresses a more stable MKP‐1 mutant protein. rIEC‐6 cells were transfected with one of three plasmids using either Fugene or Viofectin transfection reagent and a fluorescent tag (EGFP‐Cl). The three plasmids were designated either empty vector control or WT‐Flag‐tagged MKP‐1 plasmid or Flag‐tagged MKP‐1 S359/364D plasmid. The Flag MKP‐1S/D mutant carries a specific serine to aspartate mutation in the C‐terminus created using site‐directed mutagenesis. Successful clone selection was achieved with increasing concentration of G418 over a period of 14 days. Clones were grown in DMEM growth media for 24 hours. Cell lysate harvested for protein and vector expression quantified using Western blot analysis. Results demonstrated an 18× fold increase in Flag MKP‐1S/D expression compared to WT‐Flag MKP‐1 protein in rIEC‐6 cells. In conclusion, expression of a more stable MKP‐1 protein in intestinal epithelial cells will enable further study of the critical role of MKP‐1 in the intestinal inflammatory response. Support or Funding Information Liu (PI) R21 AI113930 NIH/NIAID