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Research Area
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Description
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Non-small cell lung cancer,Head and neck cancer |
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Protocol
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Kinase Assay
[1]
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| In vitro kinase activity assays |
The wild type tyrosine kinase domain of the human EGFR as well as that of the EGFR L858R/T790M double mutant are fused to Glutathione-S-transferase (GST), and extracted. Enzyme activity is then assayed in the presence of the inhibitor BIBW2992, serially diluted in 50% DMSO. A random polymer pEY (4:1) is used as substrate and biotinylated pEY (bio-pEY) is added as a tracer substrate. The kinase domain of HER2 is cloned using the baculovirus system and extracted similarly to that of EGFR kinase. Details of assays for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity are available in Supplementary information. |
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Cell Assay
[1]
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| Cell Lines |
NSCLC cells |
| Concentrations |
0-10 μM |
| Incubation Time |
1 hour |
| Methods |
1 × 104 NSCLC cells are transferred into each well of a 96-well plate and cultured overnight in serum-free media for the EGFR phosphorylation assay. After addition of BIBW2992 on the next day, the plates are incubated at 37 °C for 1 hour. EGF-stimulation is done using 100 ng/mL for 10 min at room temperature. Cells are washed with ice cold PBS, extracted with 120 μL HEPEX buffer per well and shaken at room temperature for 1 hour. In all 2 × 104 cells per well is used for the HER2 phosphorylation assay. Streptavidin precoated plates are coated with anti-EGFR-biotin at 1:100 dilution in blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Cell extracts is then transferred to the antibody-coated wells and incubated at room temperature for 1 hour. Extinction was measured at 450 nm. |
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Animal Study
[1]
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| Animal Models |
Athymic NMRI-nu/nu female mice |
| Formulation |
In 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) |
| Doses |
20 mg/kg |
| Administration |
Oral administration |
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