The remaining cell viability was measured by MTT assay

edium was discarded and dimethyl sulfoxide was added to each well to dissolve the purple formazan crystals. Plates were agitated at room temperature for 10 min and the absorbance of each 12105845 well was determined with an absorbance microplate reader at a wavelength of 570 nm. Three replicates were used for each treatment. The cell viability was determined as a percentage of the untreated control cells, by dividing the mean absorbance of each treatment by the mean absorbance of the untreated cells. EGFR MedChemExpress Mertansine expression profile in three EGFR-positive human tumor cell lines Before conducting the biological assays, we examined the EGFR expression levels in MDA-MB468, MCF-7 and Caco-2 cells, with a well-described autocrine loop of TGF-a, by flow cytometry. As represented in Fig. 3, MDA-MB-468 cells expressed the highest level of EGFR, while MCF-7 and Caco-2 cells showed similar moderate expression of EGFR. Statistical analysis Statistical analysis was performed with the SPSS statistical 17266205 software for Windows. Quantitative variables were expressed as mean and standard error. The normality of the data was tested using the Kolmogorov-Smirnov test. The differences between data with normal distribution and homogeneous variances were analyzed using the parametric Student’s t-test, otherwise the non-parametric Mann-Whitney U test was applied. A value of P,0.05 was considered significant. EGFR binding affinity of EGFt MDA-MB-468 cells were used to analyze the binding affinity of the truncated EGF analogue for EGFR. In Fig. 4, the total, specific and non-specific binding of 111In-DTPA-EGFt has been plotted against increasing concentrations of radiolabelled ligand. The Kd for specific binding of 111In-DTPA-EGFt was calculated An EGF Derivative as EGFR Blocker using a one-site binding hyperbola nonlinear regression analysis by GraphPad Prism software and indicated that the receptor-binding affinity of 111In-DTPA-EGFt for EGFR was approximately 46-fold lower than that of 111In-DTPA-hEGF. For the following experiments, we decided to use 150 nM nM of hEGF and EGFt, as a saturating concentration. In some experiments 3 nM of hEGF was also included since it is the equivalent concentration of the 150 nM EGFt, based on its binding affinity to the receptor. induced EGFR dimers, while no dimerization of EGFR could be detected after the EGFt treatment, even when the concentration was increased to 15 mM. These results suggest that the homodimers were not effectively induced or stabilized by EGFt stimulation. Moreover, when hEGF and EGFt were mixed at the same concentration, the EGFR dimer formation decreased compared to hEGF treatment, indicating that EGFt was competing with hEGF for EGFR binding and competitively decreasing dimer formation. EGFt could neither induce EGFR-HER2 heterodimerization. EGFR dimerization MDA-MB-468 cell lysates were treated with either hEGF or EGFt and in combination for 30 min and then the proteins were cross-linked by adding 40 mM of glutaraldehyde. The dimer formation was analyzed by Western blotting. As expected, hEGF EGFR activation The ability of the recombinant proteins to activate the EGFR was evaluated by analyzing tyrosine residues phosphorylation after hEGF and EGFt cell stimulation. MDA-MB-468 cells were treated An EGF Derivative as EGFR Blocker with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min at 37uC. The cell lysates were analyzed in parallel by Western blotting with an antibody against total phosphotyrosines and an antibody against EG