MTOR inhibitor and application thereof
A technology of inhibitors and proteins, applied in the direction of anti-virus agents, DNA/RNA fragments, medical preparations containing active ingredients, etc., can solve problems that have not yet been developed, and achieve a wide range of medicinal prospects and high specificity
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[0040] Example 1. A method for screening mTOR inhibitors
[0041] A method for screening mTOR inhibitors, comprising the steps of:
[0042] 1) Construct the fusion expression vector (HAFlag-xxID-Rheb) of biotin ligase (xxID) and Rheb, transfer the vector into HeLa cells, screen the HeLa-xxID-Rheb stable expression cell line, and construct the control cell line HeLa- xxID stably expressing cell lines were cultured in a medium containing 10% dialysis serum; 16 hours before the experiment, a serum-free medium was used to avoid the interference of insulin.
[0043]2) Then add insulin (0.9μM) and biotin (50μM) to the HeLa-xxID-Rheb stable expression cell line for stimulation, stimulate for 15min, add biotin (50μM) to the HeLa-xxID stable expression cell line to stimulate, without Add insulin, stimulate for 15min, lyse the sample, extract the protein, pull down with streptavidin, enrich the biotinylated protein, perform reductive alkylation on streptavidin magnetic beads, trypsin d...
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