09-310 Sigma-AldrichAnti-phospho-EGFR (Tyr1069) Antibody

Blood-brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and D-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF.

Many receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immunocytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates.