One milliliter of the pooled GPI-enriched membrane fractions defined in Table ?Table11 was immunoselected with anti-Thy-1 (A) or anti-Lck (B) antibody-coated protein A/G beads

One milliliter of the pooled GPI-enriched membrane fractions defined in Table ?Table11 was immunoselected with anti-Thy-1 (A) or anti-Lck (B) antibody-coated protein A/G beads. INTRODUCTION In contrast to transmembrane glycoproteins, surface molecules inserted into the plasma membrane via a glycosylphosphatidylinositol (GPI)1 membrane anchor are confined to the outer leaflet of the plasma membrane and do not directly communicate with the cell interior (Low, 1989 ). However, several GPI-anchored proteins have been shown to be potent transmission transducers, because their cross-linking prospects to increased protein-tyrosine phosphorylation, calcium fluxes, gene expression, and cell activation and/or proliferation (Robinson, 1991 WF 11899A ). GPI-anchored proteins such as neurotrophic factor receptors transduce signals by ligand-induced interactions with transmembrane receptor protein-tyrosine kinases (PTKs) (Massague, 1996 ), whereas other GPI-anchored molecules such as CD87 (uPAR), CD16B (FcRIIIB), and CD14 (lipopolysaccharide receptor) interact with integrins (Petty and Todd, 1996 ) WF 11899A and appear to transmission via integrin-dependent pathways. For most other GPI-anchored proteins, signaling is usually presumed to require association with sphingolipid microdomains (Romagnoli and Bron, 1997 ; Stulnig dot blot apparatus. After blocking with Tween 20CTris-buffered saline (TTBS; 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.1% Tween 20) containing 5% low-fat dry milk powder (TTBS-5% MP), the Western- and dot-blotted filters were incubated with antibodies in TTBS-1% MP for 60 min. After three washes in TTBS, the filters were Kv2.1 antibody incubated with appropriate HRP-conjugated second antibodies. After thorough washing, the filters were developed with the ECL reagent and exposed to Eastman Kodak (Rochester, NY) X-Omat S film. CT binding to membrane-associated ganglioside GM1 in the gradient fractions was evaluated by incubating the dot-blotted NC filter strips, after blocking in 5% MP-TTBS, with HRP-conjugated CT in 1% MP-TTBS at 1:20,000 dilution. After washing in TTBS the filters were developed with the ECL reagent. The luminograms were quantitated using a laser scanning densitometer (Molecular Dynamics, Sunnyvale, CA). In Vitro Kinase Assays To measure the total kinase activity, 0.5 ml of the pooled GPI-enriched membranes (fractions 3C5; corresponding to 25 106) was diluted at least fivefold with TKM buffer and centrifuged at 250,000 for 4C6 h. The sedimented membranes were suspended directly in 25 l of kinase buffer (20 mM MOPS, pH 7.4, and 10 mM MgCl2). Kinase assay was set up in a final volume of 50 l and initiated by the addition of 5 Ci of [-32P]ATP (Amersham, 5000 Ci/mmol) in 5 l of kinase buffer. For chilly kinase assays, unlabeled ATP was added to the final concentration of 50 M. In some experiments 2.5 g of acid-denatured enolase or -casein were included in the same volume of reaction mixture. To measure the kinetics of enolase phosphorylation, varying amounts of enolase were added in the same reaction volume but made up of [-32P]ATP of low specific activity (Amersham, 3 Ci/mmol). After incubation for 15 min at 25C, the reaction was halted by the addition of 6 sample buffer and boiling. Phosphorylated proteins separated in 5C20% gradient gels or 10% minigels were fixed in 10% trichloroacetic acid for 30 min and washed in 10% methanol:10% acetic acid answer for 6C12 h with several changes before drying and autoradiography. Phosphotyrosylated proteins were detected with 4G10 anti-phosphotyrosine mAb (Upstate Biotechnology, Lake Placid, NY) on Western blots. For measuring kinase activities in membrane vesicles with Thy-1 or WF 11899A Lck uncovered around the vesicle surface, 1 ml of the GPI-enriched membrane pool was diluted twofold with TKM buffer and precleared with 100 l of Pansorbin (Calbiochem) for 30C60 min at 4C with constant mixing. After the addition of anti-Thy-1 (100 l of culture supernatant) or anti-Lck (10 l) antibodies, the samples were incubated for 1 h at 4C. After adding 25 l of protein A/G-agarose beads preblocked with BSA (Santa Cruz Biotechnology), the samples were incubated for a further 4C16 h at 4C with gentle mixing. The immune complexes were sedimented, washed three times in TKM buffer and once in kinase buffer, and subjected to kinase assay as explained.