Development of an activity quantitation method for signal transduction factors in living cells using inhibitors as probes

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Some inhibitors for signal transduction factors covalently attach to the target catalytic residues in their targets only when the targets are activated and the reactivity of the catalytic residues become high. A methodology was developed that exploits this reactivity as an activity quantitation method for signal transduction factors. To detect covalent modifications by inhibitors, western blotting using antibodies raised against the modification groups as haptens was used. To evaluate the methodology, three inhibitors targeting the TOR pathway or the yeast Rim101 pathway were tested.