Neuroglobin was also colocalized with nitric oxide syn thase in the lateral teg

Moreover, overexpression of EGFR in primary tumors was associated with tumor metastasis, recurrence, and poor survival in patients with NPC. Recent data have proposed EGFR as a new target for NPC ABT-737 therapy. These studies suggest that EGFR plays a crucial role in the development and progression of NPC. EGFR is one of the most studied receptor tyrosine kinases. The natural ligands EGF and TGF a bind to the extracellular domain of EGFR, and activate the receptor and its downstream signal proteins, ultimately causing activation or modulation of various cellular processes. About 200 targets of EGFR signaling pathway have been reported, and 177 molecules involved in EGFR signaling pathway are listed in the Human Protein Reference Database but the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level.<br><br> Signaling transduction is regulated by the Adriamycin 価格 phosphory lation and dephosphorylation of proteins. Phosphopro teomics has an advantage for investigating cellular signaling pathways by simultaneously identifying a num ber of phosphoproteins at one experiment, but it also has a technical challenge because of the low abundance of phosphoproteins in cells. Therefore, enrichment of phosphoproteins is necessary before starting a phopho proteomic analysis to increase the sensitivity of identify ing phosphoproteins. Two dimensional difference gel electrophoresis is a quantitative proteomics approach with great sensitivity and accuracy of quantita tion compared to a conventional 2 DE.<br><br> Using the 2D DIGE, different samples prelabeled with ABT-199 臨床試験 mass and charge matched fluorescent cyanine dyes are co sepa rated in the same 2D gel, and an internal standard is used in every gel, overcoming the problem of intergel variation in classical 2 DE. Therefore, 2D DIGE is able to efficiently provide accurate and reproducible differen tial expression values for proteins in two or more biolo gical samples. To identify EGFR signaling proteins in NPC cells, in this study quantitative phosphoproteomics based on phosphate metal affinity chromatography enriched phosphorproteins, 2D DIGE and mass spectrometry analysis was applied to identify phosphoproteins after EGFR activation in NPC cells. We identified 33 EGFR regulated phosphoproteins, and constructed an EGFR signaling network based on the identified phos phoproteins in NPC cells.<br><br> The functional validation showed that GSTP1, one of the EGFR regulated pro teins, is involved in paclitaxel resistance in EGF stimu lated CNE2 cells. The data will provide insights into our understanding of EGFR signaling pathway and may have implications on target directed therapeutics for NPC. Methods Cell culture and EGF treatment NPC cell line CNE2 cells were cultured to 60 70% con fluency in DMEM medium supplemented with 10% fetal bovine serum at 37 C, serum starved for 24 h, and then were stimulated with 30 ngmL EGF or mock treated as a control. In EGFR blocking experiments, cells were pretreated with 1 um EGFR tyr osine kinase inhibitor PD153035, and fol lowed by incubation with EGF. Phosphoprotein enrichment A phosphoprotein purification kit was applied to enrich phosphoproteins from EGF stimulated or unstimulated CNE2 cells according to the manufac turers instructions.