Intracellular Staining by Flow Cytometry
|Detection of Phospho-EGF R/ErbB1 in EGF-treated A431 Human Cell Line by Flow Cytometry. A431 human epithelial carcinoma cell line was untreated (open histogram) or treated for 5 minutes with 100 ng/mL Recombinant Human EGF (Catalog # 236-EG, filled histogram) then stained with Human Phospho‑EGF R/ErbB1 (Y1068) Monoclonal Antibody (Catalog # MAB3570), followed by Phycoerythrin-conjugated Anti-Mouse IgG F(ab')2 Secondary Antibody (Catalog # F0102B). Mouse IgG2A (Catalog # MAB003, data not shown) was used as an isotype control. To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
|Detection of Human Phospho-EGF R/ErbB1 (Y1068) by Western Blot. Western blot shows lysates of A431 human epithelial carcinoma cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human EGF (Catalog # 236-EG) for 5 minutes. PVDF membrane was probed with 1 µg/mL of Human Phospho-EGF R/ErbB1 (Y1068) Monoclonal Antibody (Catalog # MAB3570), followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Phospho-EGF R/ErbB1 (Y1068) at approximately 190 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
Epidermal growth factor receptor (EGF R, also known as ErbB1 and HER1) is the founding member of the ErbB family of receptor tyrosine kinases. Ligand binding induces receptor dimerization and autophosphorylation on multiple tyrosine residues. Phosphorylation at Tyr 1068 allows binding of the SH2 domain of the cytosolic adaptor Grb2. This binding results in Ras activation.
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