Page 85 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy
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 amongst other events provokes Wnt and Notch activation, the former probably through stimulation of phosphoinositide-3-OH-kinase (PI3K), stimulates the activation of various pro-inflammatory signaling events and results in the activation of protein kinase A (PKA), a potential feed forward inhibitory event with respect to canonical Hedgehog transduction. Smoothened-dependent events include inhibition of PKA, stimulation of various cytoskeleton reorganizing pathways as well as stimulation of AMP-activated kinase (AMPK). In conjunction, the results provide an intellectual framework that allows understanding of context-dependent Hedgehog signaling.
Experimental procedures
Materials
Cyclopamine was from Biomol. Purmorphamine was from EMD Biochemicals (Darmstadt, Germany). Recombinant ShhN was from R&D Systems
Cell culture
Smoothened–/– fibroblasts (a gift from Dr. Taipale), Ptch1–/– and wild-type MEFs (gifts from Dr. Scott) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen). For experiments, a confluence of 50% cells was allowed to grow in six-wells plates. Stimulation were done, if appropriate with two μg/ml Shh for 10 min.
Kinome Profiling
For peptide array analysis, we employed the Pepchip kinomics array. The protocol and associated analysis has been described in detail elsewhere[55] and is based on the original protocol of van Baal et al[56]. In short, cells were washed in ice-cold PBS and lysed in a non-denaturing complete lysis buffer (cells were lysed in 50μl lysis buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5mM sodium pyrophosphate, 1mM MgCl2, 1mM glycerophosphate, 1mM Na3VO4, 1mM NaF,1μg/ml Leupeptin, 1μg/ml Aprotinin, 1mM PMSF). Subsequently the cell lysates were cleared by centrifugation and peptide array incubation mix was produced by adding 10 μl of activation mix (50% glycerol, 50 μM ATP, 0.05% v/v Brij-35, 0.25 mg/ml bovine serum albumin) and 2
                                 Noncanonical Hedgehog signaling
Noncanonical Hedgehog signalling
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