Engagement of Phospholipid Scramblase 1 in Activated Cells
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m710386200
ISSN1083-351X
AutoresDaniel Smrž, Pavel Lebduška, Lubica Dráberová, Jan Korb, Petr Dráber,
Tópico(s)Cellular transport and secretion
ResumoPhosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca2+-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcϵRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcϵRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation. Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca2+-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcϵRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcϵRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation. Phosphatidylserine (PS) 2The abbreviations used are: PS, phosphatidylserine; PLSCR1, phospholipid scramblase 1; RBL, rat basophilic leukemia; FcϵRI, high affinity IgE receptor; mAb, monoclonal antibody; αThy-1, MRCOX7 mAb specific for Thy-1.1; αFcϵRI, anti-FcϵRI α subunit-specific mAb, clone 5.14; TNP, trinitrophenyl; DNP, dinitrophenyl; pAb, polyclonal antibody; NTAL, non-T cell activation linker; LAT, linker for activation of T cells; GαM, goat anti-mouse; GαR, goat anti-rabbit; RαPLS, PLSCR1-specific rabbit pAb; αPY, phosphotyrosine-specific mAb, clone PY20; DαR, donkey anti-rabbit; Cy3, cyanine 3; MαPLS*, mouse mAb 129.2 specific for PLSCR1; FITC, fluorescein isothiocyanate; DIM, detergent-insoluble membrane(s); GST, glutathione S-transferase; BSS, buffered saline solution; BSA, bovine serum albumin; PBS, phosphate-buffered saline solution; BRA, bivariate Ripley's analysis; MαPLS, PLSCR1-specific mAb, clone TEC-23; GPI, glycosylphosphatidylinositol; Mes, 2-(N-morpholino)ethanesulfonic acid. is an anionic aminophospholipid, predominantly confined to the cytoplasmic leaflet of the plasma membrane. The appearance of PS on the cell surface is a characteristic marker of apoptotic cells and participates in the recognition and elimination by macrophages of dying, injured, senescent, or necrotic cells (1Wu Y. Tibrewal N. Birge R.B. Trends Cell Biol. 2006; 16: 189-197Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 2Brouckaert G. Kalai M. Krysko D.V. Saelens X. Vercammen D. Ndlovu M. Haegeman G. D'Herde K. Vandenabeele P. Mol. Biol. Cell. 2004; 15: 1089-1100Crossref PubMed Scopus (170) Google Scholar). In nonapoptotic cells, externalization of PS is associated with certain stages of cell development (3van den Eijnde S.M. van den Hoff M.J.B. Reutelingsperger C.P.M. van Heerde W.L. Henfling M.E.R. Vermeij-Keers C. Schutte B. Borgers M. Ramaekers F.C.S. J. Cell Sci. 2001; 114: 3631-3642Crossref PubMed Google Scholar), cell fusion (4Kenis H. van Genderen H. Bennaghmouch A. Rinia H.A. Frederik P. Narula J. Hofstra L. Reutelingsperger C.P.M. J. Biol. 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Immunol. 2002; 38: 1235-1238Crossref PubMed Scopus (12) Google Scholar) suggested that phosphorylation of this protein might be essential for the changes in the distribution of phospholipids in the plasma membrane during cell activation and degranulation. It was, however, unclear whether tyrosine phosphorylation of PLSCR1 in mast cells is confined to FcϵRI triggering and in what way it is related to PS externalization and exocytosis observed in activated mast cells. To address these issues, we studied the interrelationships among tyrosine phosphorylation of PLSCR1 and its plasma membrane topography, PS externalization, and degranulation in RBL cells triggered by various stimuli known to have different effects on protein tyrosine phosphorylation, [Ca2+]i, and exocytosis. Our data indicate that the above events are independent phenomena, which can occur individually, depending on the activation methods employed. Antibodies and Reagents—The following antibodies were used: MRCOX7 monoclonal antibody (mAb) specific for Thy-1.1 (αThy-1) (32Mason D.W. Williams A.F. Biochem. J. 1980; 187: 1-20Crossref PubMed Scopus (446) Google Scholar), anti-FcϵRI α subunit-specific mAb, clone 5.14 (αFcϵRI) (33Baniyash M. Alkalay I. Eshhar Z. J. Immunol. 1987; 138: 2999-3004PubMed Google Scholar), anti-FcϵRI-β subunit-specific mAb (34Rivera J. Kinet J.-P. Kim J. Pucillo C. Metzger H. Mol. Immunol. 1988; 25: 647-661Crossref PubMed Scopus (79) Google Scholar), trinitrophenyl (TNP)-specific IgE mAb (IGEL b4 1) (35Rudolph A.K. Burrows P.D. Wabl M.R. Eur. J. Immunol. 1981; 11: 527-529Crossref PubMed Scopus (174) Google Scholar), dinitrophenyl (DNP)-specific IgE mAb (36Liu F.-T. Bohn J.W. Ferry E.L. Yamanoto H. Molinaro C.A. Sherman L.A. Klinman N.R. Katz D.H. J. Immunol. 1980; 124: 2728-2737PubMed Google Scholar), rabbit polyclonal antibody (pAb) specific for non-T cell activation linker (NTAL) and linker for activation of T cells (LAT) (37Dráberová L. Shaik G.M. Volná P. Heneberg P. Tůmová M. Lebduška P. Korb J. Dráber P. J. Immunol. 2007; 179: 5169-5180Crossref PubMed Scopus (23) Google Scholar), goat anti-mouse (GαM) IgG-10 nm, and goat anti-rabbit (GαR) IgG-5 nm gold particle conjugates (Amersham Biosciences). PLSCR1-specific rabbit pAb (RαPLS) was prepared by immunizing rabbits with recombinant PLSCR1. Phosphotyrosine-specific mAb, clone PY20 (αPY), conjugated to horseradish peroxidase, was purchased from BD Biosciences. Horseradish peroxidase-conjugated GαM IgG and horseradish peroxidasee-conjugated GαR IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Donkey anti-rabbit (DαR) IgG-cyanine 3 (Cy3) conjugate was bought from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Mouse mAb 129.2 specific for PLSCR1 (MαPLS*) was kindly provided by M. Benhamou (INSERM, Institut Pasteur, Paris, France). Annexin V-fluorescein isothiocyanate (FITC) was from BD Biosciences. Latrunculin B, EGTA, phenylmethylsulfonyl fluoride, calcium ionophore A23187, and all other chemicals were from Sigma. Production of New mAb against Plasma Membrane Microdomains and Identification of Their Target Antigens—RBL cells were lysed in ice-cold 1% Brij 96-containing lysis buffer (38Surviladze Z. Dráberová L. Kovářová M. Boubelík M. Dráber P. Eur. J. Immunol. 2001; 31: 1-10Crossref PubMed Scopus (45) Google Scholar). The lysate was subjected to fractionation on a sucrose density gradient, as described previously (38Surviladze Z. Dráberová L. Kovářová M. Boubelík M. Dráber P. Eur. J. Immunol. 2001; 31: 1-10Crossref PubMed Scopus (45) Google Scholar), and low density fraction, containing detergent-insoluble membranes (DIM), were pooled and used for immunization of BALB/c mice. Hybridoma cells were obtained after fusion of SPO2 mouse myeloma cells with spleen cells of immunized mice using standard procedures (39Dráber P. Zikán J. Vojtíšková M. J. Immunogenet. 1980; 7: 455-474Crossref PubMed Scopus (34) Google Scholar). The target antigens of the new mAb were immunoprecipitated from the lysate of resting RBL cells and separated by two-dimensional gel electrophoresis under reducing conditions. Alignment of immunoblotted and colloid silver-stained membranes allowed identification of silver-stained proteins corresponding to the target antigens. The corresponding in-gel silver-stained proteins were excised and destained as described previously (40Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (842) Google Scholar). The proteins were digested by trypsin and analyzed by peptide mass mapping and/or peptide sequencing (41Bobek J. Halada P. Angelis J. Vohradský J. Mikulík K. Proteomics. 2004; 4: 3864-3880Crossref PubMed Scopus (28) Google Scholar). Data base searches of the identified peptide fragments allowed determination of the target antigens. DNA Constructs, Recombinant Proteins, and Immunoaffinity Purification of pAb—To prepare the N-terminal fragment of recombinant PLSCR1, full-length cDNA of rat PLSCR1 without initial ATG codon was amplified and cloned into XmaI and HindIII cloning sites of pQE30 expression vector (Qiagen, Hilden, Germany) using the forward (5′-AAACCCGGGGAGAAGCACGGACCACCAGAA-3′) and reverse (5′-CCCAAGCTTGCTACCATACTCCTGACCTTTG-3′) primers. A DNA fragment of the cloned cDNA was excised by SpHI and BamHI restriction enzymes and cloned into the corresponding sites in pQE70 expression vector (Qiagen). To prepare the glutathione S-transferase (GST)-PLSCR1 fusion protein, full-length cDNA of rat PLSCR1 without the initial ATG codon was cloned into EcoRI site of pGEX-3X expression vector (Amersham Biosciences) using the following primers: forward, 5′-AAGGAATTCCGAGAAGCACGGACCACCAGAA-3′; reverse, 5′-AAAGAATTCCTACCATACTCCTGACCTTTG-3′. Recombinant proteins were expressed in bacteria Escherichia coli strain JM109. The N-terminal fragment of PLSCR1 was isolated from inclusion bodies as previously reported (42Konvalinka J. Heuser A.M. Hrušková-Heidingsfeldová O. Vogt V.M. Sedláček J. Štrop P. Kräusslich H.G. Eur. J. Biochem. 1995; 228: 191-198Crossref PubMed Scopus (38) Google Scholar) and used to immunize rabbits to generate RαPLS. The recombinant GST-PLSCR1 was affinity-purified on glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's protocol. The isolated GST-PLSCR1 or recombinant fragment of rat NTAL (37Dráberová L. Shaik G.M. Volná P. Heneberg P. Tůmová M. Lebduška P. Korb J. Dráber P. J. Immunol. 2007; 179: 5169-5180Crossref PubMed Scopus (23) Google Scholar) was covalently bound to CNBr-activated beads (Sigma), which were then used for immunoaffinity purification of PLSCR1- or NTAL-specific pAb (37Dráberová L. Shaik G.M. Volná P. Heneberg P. Tůmová M. Lebduška P. Korb J. Dráber P. J. Immunol. 2007; 179: 5169-5180Crossref PubMed Scopus (23) Google Scholar). In some experiments, isolated GST-PLSCR1 fusion protein was used at a concentration of 50 μg/ml to confirm the specificity of anti-PLSCR1 antibodies. Cells and Their Activation—The origin of RBL cells, clone 2H3, and their culture conditions have been described (43Siraganian R.P. McGivney A. Barsumian E.L. Crews F.T. Hirata F. Axelrod J. Fed. Proc. 1982; 41: 30-34PubMed Google Scholar, 44Dráberová L. Dráber P. Eur. J. Immunol. 1991; 21: 1583-1590Crossref PubMed Scopus (29) Google Scholar). Before activation, cells were harvested and washed with buffered saline solution (BSS; 20 mm HEPES, pH 7.4, 135 mm NaCl, 5 mm KCl, 1 mm MgCl2, 5.6 mm glucose), supplemented with 1.8 mm CaCl2 and 0.1% bovine serum albumin (BSA). The cells were sensitized or not with TNP-specific IgE (IGEL b4 1 ascitic fluid diluted 1:1000 in BSS/BSA), αFcϵRI, or biotin-labeled αThy-1 (both at 2 μg/ml), washed, and resuspended in BSS/BSA to 20 × 106/ml. The cells were activated at 37 °C for the indicated time intervals by adding equal amounts of twice concentrated activators in BSS/BSA. Final concentrations of the activators were as follows: TNP-BSA (1 μg/ml), αFcϵRI or αThy-1 (2 μg/ml), streptavidin (10 μg/ml), calcium ionophore A23187 (3 μm), and pervanadate (0.2 mm sodium orthovanadate, 1 mm hydrogen peroxide). To induce apoptosis by UV irradiation, cells grown in medium were exposed in open Petri dishes to UV-C (predominantly 254 nm) from a germicidal lamp (Philips TUV G30T8 30-W bulb) at 60 cm distance. After a 10-min exposure, fresh medium was added, and the cells were cultured for further 6 h before analysis. Apoptosis was confirmed by proteolytic cleavage of Lyn (45Luciano F. Ricci J.E. Auberger P. Oncogene. 2001; 20: 4935-4941Crossref PubMed Scopus (54) Google Scholar) and DNA laddering as previously described (46Loo D.T. Rillema J.R. Methods Cell Biol. 1998; 57: 251-264Crossref PubMed Google Scholar). Sucrose Density Gradient Fractionation—Cells were lysed in ice-cold Brij 58 lysis buffer (25 mm Mes, pH 6.5, 100 mm NaCl, 2 mm EDTA, 0.5% Brij 58, phosphatase inhibitors (2 mm Na3VO4 and 10 mm glycerolphosphate), 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). After 30 min, the lysate was subjected to fractionation on a sucrose density gradient (38Surviladze Z. Dráberová L. Kovářová M. Boubelík M. Dráber P. Eur. J. Immunol. 2001; 31: 1-10Crossref PubMed Scopus (45) Google Scholar) supplemented with 2 mm Na3VO4 and 10 mm glycerolphosphate. Determination of Cell Degranulation and PS Externalization—Degranulation of the cells and PS externalization were assessed by the amount of released β-glucuronidase and bound FITC-labeled annexin V, respectively, as described (9Smrž D. Dráberová L. Dráber P. J. Biol. Chem. 2007; 282: 10487-10497Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Immunoprecipitation, Immunoblotting, and Silver Staining—Toward the end of the activation period, cells (107) were briefly centrifuged, and the pellet was lysed in ice-cold Triton X-100 lysis buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 2 mm EDTA, 1% Triton X-100, 2 mm Na3VO4, 10 mm glycerol phosphate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). After 20 min, the lysate was centrifuged at 16,000 × g for 5 min at 4 °C, and the supernatant was further processed. To immunoprecipitate antigens recognized by mouse mAb, beads with covalently bound anti-mouse IgG (Sigma) were incubated with hybridoma supernatant for 2 h at 4 °C. The beads were washed with lysis buffer and incubated with cell lysate for 2 h at 4 °C. The immunoprecipitated antigen was eluted with glycine buffer (50 mm glycine-HCl, pH 2.5, 100 mm NaCl). To immunoprecipitate PLSCR1, a 1-ml aliquot of the cell lysate was incubated with 15 μl of serum from PLSCR1-immunized rabbits for 2 h at 4 °C, followed by incubation with UltraLink-immobilized protein A (Pierce) for 2 h at 4 °C. Alternatively, 1 ml of the cell lysate was incubated with 10 μg of immunoaffinity-purified RαPLS or 2 μg of affinity-purified MαPLS*. Immunoprecipitated proteins were eluted by boiling in reducing Laemmli sample buffer for 10 min. The samples were analyzed by immunoblotting of SDS-PAGE-fractionated proteins or by two-dimensional electrophoresis, as described previously (47Schraven B. Ratnofsky S. Gaumont Y. Lindegger H. Kirchgessner H. Bruyns E. Moebius U. Meuer S.C. J. Exp. Med. 1994; 180: 897-906Crossref PubMed Scopus (17) Google Scholar). Immunoblotting was performed with 2 μg/ml purified antibodies, or 1:1000 diluted sera or ascitic fluids. Horseradish peroxidase-conjugated αPY, GαM, or GαR IgG were diluted 1:10,000. Horseradish peroxidase-generated signal was detected by enhanced chemiluminescence (Amersham Biosciences) on x-ray film and quantified by Luminescent Image Analyzer LAS-3000 (Fuji Photo Film Co., Tokyo). SDS-PAGE or two-dimensional fractionated proteins were visualized by in-gel silver staining using silver nitrate in combination with formaldehyde developer in alkaline carbonate buffer (48Patton W.F. J. Chromatogr. B. 2002; 771: 3-31Crossref PubMed Scopus (419) Google Scholar, 49Merril C.R. Goldman D. Sedman S.A. Ebert M.H. Science. 1981; 211: 1437-1438Crossref PubMed Scopus (2106) Google Scholar) or by membrane colloid silver staining (50Vettermann C. Jäck H.M. Mielenz D. Anal. Biochem. 2002; 308: 381-387Crossref PubMed Scopus (20) Google Scholar). Confocal Microscopy—Cells were spun down and resuspended in annexin V binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) at a concentration of 5 × 106 cells/ml. Forty μl of the cell suspension was mixed with 3 μl of annexin V-FITC and incubated at room temperature in the dark for 15 min. The cells were then transferred on poly-l-lysine-coated coverslips (12 mm in diameter) placed in wells of a 24-well plate. Five min later, the cells were fixed with 5% paraformaldehyde in annexin V binding buffer for 30 min, washed with phosphate-buffered saline solution, pH 7.2 (PBS), and nonspecific binding sites were blocked for 10 min with PBS supplemented with 5% BSA (PBS/BSA; also used for dilution of antibodies). After a 5-min permeabilization with 0.1% Triton X-100 in PBS and washing, the cells were incubated for 20 min with PBS/BSA and then labeled for 1 h with immunoaffinity-purified RαPLS (5 μg/ml) or rabbit anti-LAT serum (diluted 1:1000), followed by washing with PBS, blocking for 20 min with PBS/BSA, and incubation for 1 h with DαR IgG-Cy3 conjugate (10 μg/ml). After another washing step, the cells were fixed for 10 min with 5% paraformaldehyde in PBS, washed, and mounted in p-phenylenediamine (1 mg/ml, 50% glycerol in PBS). Images were acquired with a Leica TCS NT/SP confocal system in conjunction with Leica DMR microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with oil objective ×100/1.4 numerical aperture. Electron Microscopy—Plasma membrane sheets were prepared as described (51Sanan D.A. Anderson R.G.W. J. Histochem. Cytochem. 1991; 39: 1017-1024Crossref PubMed Scopus (87) Google Scholar, 52Wilson B.S. Steinberg S.L. Liederman K. Pfeiffer J.R. Surviladze Z. Zhang J. Samelson L.E. Yang L. Kotula P.G. Oliver J.M. Mol. Biol. Cell. 2004; 15: 2580-2592Crossref PubMed Scopus (179) Google Scholar). Briefly, cells in complete culture medium were grown overnight on glass coverslips in the presence or absence of DNP-specific IgE (1 μg/ml). Adherent cells were activated or not by DNP-BSA (1 μg/ml) or other activators in BSS/BSA for the indicated time intervals at 37 °C, and the glass coverslips with cells were washed in ice-cold PBS and then inverted and briefly pressed onto pioloform-covered and poly-l-lysine-coated electron microscopy grids kept on ice; careful separation of coverslips from the grids leaves the plasma membrane sheets attached to the grids. The membranes were fixed immediately with 2% paraformaldehyde and then exposed to immunoaffinity-purified RαPLS (10 μg/ml), rabbit anti-NTAL pAb (10 μg/ml), or anti-FcϵRI-β mAb (3 μg/ml). Washed membranes were exposed to gold-conjugated secondary antibodies diluted 1:20 from commercial stocks. After additional fixation with 2% glutaraldehyde and staining with osmium tetroxide, tannic acid, and uranyl acetate, the samples were dried and examined under a JEOL JEM 1200EX electron microscope operating at 60 kV. To obtain membrane sheets from cells with aggregated Thy-1, the cells were incubated for 15 min at room temperature with αThy-1 (1 μg/ml), washed, and then incubated at 37 °C for 10 min with GαM IgG-10-nm gold conjugate. In the case of Thy-1 labeling without aggregation, the cells were first fixed with paraformaldehyde and labeled for 15 min with αThy-1, followed by 10-min exposure to GαM IgG-10-nm gold conjugate. After the extracellular labeling, plasma membrane sheets were isolated as described above. Plasma membrane sheets from nonadherent apoptotic cells were prepared as described (53Lebduška P. Korb J. Tůmová M. Heneberg P. Dráber P. J. Immunol. Methods. 2007; 20: 139-151Crossref Scopus (14) Google Scholar). Evaluation of the Results—Means ± S.D. were computed from at least three independent experiments. Statistical significance of intergroup differences was calculated using an unpaired Student's t test; each group consisted of four independent experiments. Evaluation of gold particle co-localization was performed by bivariate Ripley's analysis (BRA) using the L(r)-r function as described (52Wilson B.S. Steinberg S.L. Liederman K. Pfeiffer J.R. Surviladze Z. Zhang J. Samelson L.E. Yang L. Kotula P.G. Oliver J.M. Mol. Biol. Cell. 2004; 15: 2580-2592Crossref PubMed Scopus (179) Google Scholar). Pictures of plasma membrane sheets from two independent experiments were taken to cover ∼50 μm2 of plasma membrane surface and analyzed using Matlab software algorithms (52Wilson B.S. Steinberg S.L. Liederman K. Pfeiffer J.R. Surviladze Z. Zhang J. Samelson L.E. Yang L. Kotula P.G. Oliver J.M. Mol. Biol. Cell. 2004; 15: 2580-2592Crossref PubMed Scopus (179) Google Scholar). Significant co-localization (p < 0.01) of the proteins occurred when the position of the L value curve (solid lines) surpassed the boundaries (dashed lines) predicted for random distribution of gold particles at a corresponding distance (Fig. 3, E and F and Fig. 5, D, and E). Cluster size was determined by the program GOLD (54Philimonenko A.A. Janáček J. Hozák P. J. Struct. Biol. 2000; 132: 201-210Crossref PubMed Scopus (71) Google Scholar).FIGURE 5Properties of PLSCR1 in cells activated through extensively aggregated Thy-1. A, tyrosine phosphorylation of PLSCR1 after Thy-1 aggregation. Cells were sensitized or not (C-) for 30 min on ice with biotinylated αThy-1. The cells were washed and activated at 37 °C with streptavidin (αThy-1/Streptavidin) for the time intervals indicated. Cell lysates were analyzed by immunoblotting (IB) for tyrosine-phosphorylated proteins with αPY. PLSCR1 was immunoprecipitated (IP) from the same cell lysates with RαPLS and analyzed by immunoblotting with αPY. The amount of immunoprecipitated PLSCR1 was det
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