ADAM17 over expression in AGS gastric epithelial cells

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As H. pylori stimulated the highest ADAM17 levels in the AGS gastric epithelial cell line (Figure 1A), AGS cells were chosen for constructing ADAM17 over expression and C-terminus deletion mutants. ADAM 17 has three phosphorylation sites located within its C-terminus. To analyse the effects of H. pylori on ADAM17 activity and its C-terminal phosphorylation at T735, S791 and S819, AGS gastric epithelial cells over expressing both wild type and C-terminus deficient ADAM17 were constructed. The ΔC-terminus ADAM17 mutant had a translational stop signal introduced resulting in the absence of the cytoplasmic tail and T735, S791 and S819 phosphorylation sites. Protein expression from plasmid constructs was controlled via a Tet-off system, which allowed for inducible over expression of ADAM17 in the absence of tetracycline. ADAM17 over expressing monoclones were selected and over expression validated using reverse transcription followed by PCR and immunofluorescence microscopy. Over expressed ADAM17 had the same cellular localisation profile as the endogenous ADAM17 and no compartmentalisation was observed. Due to the very high efficiency of ADAM17 overexpression we hypothesise that in these cells overexpressed ADAM17 will be the dominant clone and the biological driver.