The cells were incubated with 10 nM of EGF for 1, 5, 10, 30, 120 and 360 minutes and then washed two times with phosphate buffered saline and lysed with Bio Plex lysis buffer. Cell lysates were cleared by Volasertib aml centrifugation, and the total pro tein concentration of the supernatant was determined using a protein assay reagent and analyzed by western blot. Cells that were not treated with growth hormone were used as the control. Western blot analysis SDS PAGE and membrane transfer were performed using standard protocols. Antibodies against anti phos pho EGFR, doubly phosphorylated p44 42 ERK, ERK, phospho MEK1 2, MEK, Mig6 and actin were purchased from Cell Signaling Technology, Inc. Anti phospho Shc, anti Shc antibodies and anti EGFR antibodies were purchased from Upstate Biotechnology.

Protein band intensities were Inhibitors,Modulators,Libraries quantified using a densitometer. Normalization procedure is described in earlier study. Briefly, the maximum value of protein phosphorylation level among three cell lines was set to 1 and the values at t 0 min utes were set to 0 under the assumption that all the proteins were inactive before EGF stimulation. We con sidered that total protein level of EGFR is equal in EGFR WT and L858R cells. All concentrations of Shc, MEK and ERK were considered to be equal in three cell lines. Mig6 overexpression The MIG6 gene was amplified from a human ERRFI1 cDNA purchased from OriGene using the primers. The resulting DNA fragment was cloned into the vector pCMV 6 Neo using the Xba I restriction site. Cells were seeded in 96 well plates at Inhibitors,Modulators,Libraries 1 105 cells well.

Transfec tion of the MIG6 gene was performed using the Lipofec tamine LTX and CombiMAG magnetofection kit according to manufacturers protocol. Control cells were transfected with pCMV 6 Neo vector. After 8 hours of transfection, cells were supplemented Inhibitors,Modulators,Libraries with serum free RPMI1640 media. The following day, cells were treated with 10 nM EGF in the presence or absence Inhibitors,Modulators,Libraries of gefitinib. Cell Viability Assay Cell viabilities of H1299 cells were measured by an MTT cell proliferation assay 3 days after stimu lation with or without 10 nM EGF in the presence of various doses of gefitinib using the Cell Count Kit SF. The cell viability was determined by optical density at 450 nm. Computation To model EGFR signaling network, we adopted a deter ministic ordinary differential equation model.

Model scheme is described in Additional file 1, Table S1 5. Additional file 1, Table S1 and 2 summarize the biochemical reactions with 29 components and 27 dif ferential equations, which Inhibitors,Modulators,Libraries are given by mass action or Michaelis selleck chemical Menten kinetics. Additional file 1, Table S3 and 4 list the parameter values and the initial concentra tions of the cellular signaling molecules. These values were estimated based on the parameter ranges which were listed in Additional file 1, Table S5. Our pathway network is drawn by Cell Designer 4.