Page 90 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

FIGURE 1. Outline of the study. A. Technical approach – kinome profiling. In this study, we aim to comprehensive characterize cellular kinase enzymatic activities. To this end appropriately stimulated cell cultures are washed with ice-cold PBS and lysed in a non-denaturing complete lysis buffer as to solubilize cellular kinases. Lysates are the transferred to arrays consisting of a substrate peptide library, spotted in triplicate as to assess technical reproducibility, which are spotted on a hydrogel-coated glass carrier. Upon addition of radioactive ATP and an activation mix, kinases –if enzymatically active- will phosphorylate substrate peptides. Incorporation of radioactive ATP into a substrate peptide is taken as a measure of enzymatic kinase activity towards a particular substrate. The broad variation in specific substrates used (see also supplementary data) allows obtaining a more-or-less complete description of cellular signaling, the so-called kinome. B. Biological approach. In this study, we first generate a description of the effects of Shh challenge on cellular signaling in general by comparing kinome profiling results of cultures challenged and not challenged by the morphogen. To delineate the contribution of possible non-Ptc- dependent components to Hedgehog-dependent kinase events, the effect of Shh stimulation on fibroblast genetically devoid of Ptc is established as well. To identify signal transduction events that are downstream of Ptc but do not involve Smo, the Hedgehog provoked effects on the cellular kinome are studied in fibroblasts genetically deficient for Smo. Finally, to identify events that are solely dependent on the activation of Smo, we study the effects of the Smo agonist purmorphamine
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