Page 87 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

using this dichotomal testing based on number of Markov state “on” peptides did not result in statistical significance, the relative levels of phosphorylation were also tested using a paired T test, directly parametrically comparing phosphorylation of the corresponding spots. As we considered thus-discovered statistically significant differences between the relevant experimental conditions less robust, in the depiction of the results they have been highlighted with an orange border. Note that due to differences in the number of peptides allotted to the signal transduction categories apparently large differences in phosphorylation not always yield statistically significant results, while smaller differences can produce such results if the number of substrates in such categories is large.
Results and discussion
Hedgehog stimulation provoked rapid and marked reorganization of the cellular kinome
The general approach as to this study, both technically and biologically is provide through Figure 1. We characterised the kinase signatures associated with Hedgehog stimulation of mouse embryonic fibroblasts (MEFs), which we have recently shown to constitute a powerful model for delineating signal transduction events[39]. We established that under our experimental conditions, these cells do not endogenously release Hedgehog (not shown). Cells were incubated for 10 min with either 1 μg/ml Shh or a vehicle control, and the cell lysates were employed for in vitro phosphorylation of peptide arrays using 33P-γ-ATP. Arrays consisted of 1024 different undecapeptides, of which 48 are various technical controls, whereas the remaining 976 peptides provide kinase substrate consensus sequences spanning the entire mammalian kinome and which we have shown earlier to provide comprehensive insight in cellular signal transduction[40]. On each separate carrier, the array was spotted three times, to allow assessment of possible variability in substrate phosphorylation. The final physical dimensions of the array were 25 x 75 mm, each peptide spot having a diameter of approximately 250 μm, and peptide spots being 620 μm apart. As a control for the specificity of the reaction 33P--ATP was used; no incorporation of radioactivity was seen. We then calculated the mean phosphorylation level for all substrates before and after the treatment (total number of data points is 9 for each group). The technical quality of the profiles was good, and we only allowed experiments in which the Pearson product moment correlation coefficient was more as 0.95 for the technical replicas. We decided to use these data
Noncanonical Hedgehog signaling
Noncanonical Hedgehog signalling
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