[30]][“Inhibitor D”

Surprisingly, bombesinst imu la tes tyro sine phosphoryFluphenazine hydrochloride lation through a PKC independentpathway.The integrity of thepolymerized actin network is essential for the function of both PKCdependent and independent pathways of tyros ine phosphorylation. S tud ieswi th human umbilical vein endo the followed by transendothelial migration into the brain lialcells in culture have suggested th at leukocyte function parenchyma.These findings demon larcell adhes ion molecule in intracellu larsignaling.For this purpose, we used im mortalizedratbr ain microvessel endo thelial cells, which exh ibit no app arentmorphologictransfo rmation and re tain in cultu re the diffe rentia ted pheno type of endo thelialcells and seve ralst ruc tu ral and pharmacological charac ter istics of thebloodbrain barr ier. Ourre sults indic ate th atcrosslinking of I C A induces tyros ine phosphorylation of several pro te ins toge ther withst imu lation of p activity in RBE cells.Extrava sation of lymphocytes in to per ivascu lar tissue occurs during no rmal recircu lation as well as dur ing infil tration in to si tesof inf lammation. Th is process requ ires adhes ion to, fol lowed bymigration between, vascu lar endo thelialcells.Several families of adhes ion molecules have been identified th atpar tic ipa te in the in te ractionsbetween lymphocytes and endo the lialcells. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby Albamycinsodium marked advertisement in accordance with U.The abbreviations used are: ICA, intercellular adhesion mol ecule l; RAM, rabbit antimouse immunoglobulins; RBE, rat brain en dothelium; PAGE, polyacrylamide gel electrophoresis.Cells were then incuba ted for min at C,wi th antiICAM antibody; af ter pgml; I, anti CA lantibody, crosslinked with RAM, cross wash ings, RAM antibod ies were added for min, un less linked with RAM.Followingtheincubations,thecells were lysedwi th otherw ise ind ica ted, for ICAM crosslinking.Cells were washed be SDS samp le buffer and ana lyzed for phosphotyrosyl containing proteins fore lysis.Insome experiments, antibody trea tment was rep laced by by immunoblot.Positions of mo lecu lar mass markers are indi incubation with PAS cells for min at C.SDS PAGE and immunob lott ings we re then perfo rmed as previously de scr ibed. Blots were probedwi th polyclonal anti bodies aga inst phosphatidylinosito l kinase and, after seve ral washes, wi th appropr ia te peroxidasecoupled antibod ies. The kinase activity of the immunop rec ipita tes was de te rm ined us ing eno la se as exogenous substrate. The presence of phosphorylated proteins was revealed by au torad iography ofthenitrocellulose membrane.The level of immuno precipitated p was then ana lyzed by immunob lott ing us ing anti p antibody and pe rox ida se coupled sheep antimouse I gG. ECL reagents we re used for de tection of peroxidase ac tivity.Tyrosine phosphoryla tion was assessed by ana lyz ing RBE cell lysa tes by immuno blott ing wi th antiphosphotyrosine antibody.The specificity of the response was further confirmed by competitionwi th an excess of phos phoamino acid; phosphotyrosine, but neitherphospho threon ine nor phosphoserine, completely abolished p label ing.

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