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In vitro EGFR kinase assay

FG Feng Gao
XY Xinfang Yu
ML Ming Li
LZ Li Zhou
WL Wenbin Liu
WL Wei Li
HL Haidan Liu
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The recombinant active WT EGFR, L858R EGFR, L858R/T790M EGFR, and Del E746-A750 EGFR were purchased from Millipore. The in vitro EGFR kinase assay was performed as described previously25. Briefly, active EGFR (100 ng) was mixed with various doses of deguelin or 100 nM gefitinib. The reaction was incubated with 500 μM angiotensin II for 5 min at room temperature, followed by incubation with 10 μL of ATP mixture (25 mM MgAc and 0.25 μM ATP containing 10 μCi [γ-32P] ATP) for 15 min at 30 °C and then 25 μL of reaction mixture was transferred onto P81 papers. The papers were washed with 0.75% phosphoric acid twice and then with acetone once. The radioactive incorporation was determined using a scintillation counter.

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