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Inhibition of EGFR kinase activity and autophosphorylation by Iressa.
Inhibition of EGFR kinase activity and autophosphorylation by Iressa. Inhibition of EGFR autophosphorylation on Tyr1173 by Iressa in Hec50co cells (A) and Ishikawa H cells (B). Cells were serum starved for 24 h and treated as indicated with 30 ng/mL EGF for 15 min with increasing concentrations of Iressa. Lane 1, untreated control; lane 2, cells treated with EGF; lane 3, cells treated with 1 μL DMSO (vehicle) and EGF; lane 4, cells treated with 40 μL DMSO (vehicle) and EGF; lane 5, cells treated with 0.1 μmol/L Iressa and EGF; lane 6, cells treated with 1 μmol/L Iressa and EGF; lane 7, cells treated with 5 μmol/L Iressa and EGF; lane 8, cells treated with 10 μmol/L Iressa and EGF; lane 9, cells treated with 20 μmol/L Iressa and EGF; lane 10, cells treated with 40 μmol/L Iressa and EGF. Iressa at all concentrations blocked EGFR autophosphorylation on Tyr1173. DMSO (1 μL) serves as a vehicle control for the cells treated with 0.1 μmol/L Iressa (lane 5), whereas lane 4 included a vehicle control of DMSO volume of 40 μL to compare to lane 10, where 40 μL Iressa in DMSO was used (total concentration of Iressa, 40 μmol/L). Enhanced phosphorylation of EGFR was observed in lane 4 (B) in the presence of a larger volume of DMSO vehicle for Hec50co cells. Nevertheless, this was completely blocked in the presence of Iressa as shown in lane 10. C, in vitro kinase assay of EGFR in Hec50co cells. Cells were treated as indicated with EGF, Iressa, or both after serum starvation for 24 h. Iressa (1 μmol/L) for 2 h blocked EGFR kinase activity and autophosphorylation on tyrosine residue Tyr1173. D, total EGFR in Ishikawa H and Hec50co cells. A comparable amount of EGFR total protein is present in the two cell lines. Lina Albitar et al. Mol Cancer Ther 2005;4: ©2005 by American Association for Cancer Research
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