Article |
Address correspondence to Amnon Altman or Martin Villalba, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: (858) 558-3527. Fax: (858) 558-3526. E-mail: amnon{at}liai.org or Villalba{at}liai.org
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Abstract |
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Key Words: protein kinase C-; phospholipase C; Vav; phosphatidylinositol 3-kinase; T cell
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Introduction |
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The selective mechanism that recruits PKC to the SMAC/IS during antigen stimulation remains elusive. In this regard, we found recently that Vav and Rac selectively promote the membrane and cytoskeleton translocation of PKC
, and mediate its enzymatic activation by CD3/CD28 costimulation in a process that depends on actin cytoskeleton reorganization (Villalba et al., 2000a). A similar pathway mediates the antigen-induced translocation of PKC
into lipid rafts (Bi et al., 2001; Villalba et al., 2001). Similarly, recent reports indicate functional cooperation between Vav and PKC
in several T cell signaling pathways (Dienz et al., 2000; Hehner et al., 2000; Moller et al., 2001) and with the finding that Vav is essential for actin polymerization and TCR cap formation after TCR/CD3 ligation (Fischer et al., 1998; Holsinger et al., 1998; Wülfing et al., 2000). Because this effect was specific for PKC
(Villalba et al., 2000a), we hypothesized that it may represent a novel mechanism, which is independent on the conventional PKC activation pathway mediated by phospholipase C-
1 (PLC
1). In this pathway, TCR-mediated tyrosine phosphorylation and subsequent activation of PLC
1 (Granja et al., 1991; Park et al., 1991; Secrist et al., 1991; Weiss et al., 1991) lead to hydrolysis of inositol phospholipids and production of the second messenger, DAG. Membrane-associated DAG is an essential cofactor that binds, recruits, and subsequently activates Ca2+-dependent conventional PKCs (cPKCs) and Ca2+-independent novel PKCs (nPKCs) in the plasma membrane (Nishizuka, 1995; Irvin et al., 2000; Zhang et al., 2000). PLC
1 plays an important role in T cell activation, as T cells expressing a LAT mutant, which cannot recruit and activate PLC
1, are deficient in several downstream signaling events, including Ca2+ mobilization and activation of the Ras/ERK pathway and NFAT (Irvin et al., 2000; Zhang et al., 2000). Similarly, a PLC
1-deficient T cell line was recently found to display severe activation defects (Irvin et al., 2000).
In the present work, we examined the role of PLC1 in the membrane and lipid raft recruitment of PKC
and its catalytic activation in T cells. Using three independent approaches to deplete or inhibit cellular PLC
1 activity, we demonstrate that the membrane recruitment and activation of PKC
(but not PKC
) are independent of PLC
1. We further show that this mechanism involves Vav, phosphatidylinositol 3-kinase (PI3-K), and, indirectly, 3-phosphoinositide-dependent kinase-1 (PDK1). These results support the existence of a novel mechanism, which plays a role in the selective TCR-induced activation of PKC
and, potentially, its recruitment to the T cell synapse.
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Results |
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Next, we compared the receptor-induced membrane translocation of PKC or PKC
in J.
1, a PLC
1-deficient cell line, versus J.
1.WT-2, a PLC
1-reconstituted cell line derived from this mutant (Irvin et al., 2000). In the J.
1 cells, anti-CD3 plus anti-CD28 stimulation still induced PKC
, but not PKC
, translocation (Fig. 1 C). Reconstitution of J.
1 cells with wild-type PLC
1 (J.
1.WT-2) restored PKC
translocation, with a minimal effect on PKC
translocation. Calculation of the PKC membrane/cytosol expression ratio for each group of cells makes it evident that: (a) Stimulation increases the relative membrane expression of both PKC
and
in the PLC
1-reconstituted cells; and (b) In the PLC
1-deficient cells, stimulation still increases the relative membrane expression of PKC
, but not PKC
.
Anti-CD3/CD28 stimulation induces a Vav/Rac-dependent (Villalba et al., 2001) PKC translocation to membrane lipid rafts, which also localize at the IS (Bi et al., 2001). Therefore, we wished to determine whether this lipid raft translocation of PKC
requires PLC
1. Detergent-insoluble glycolipid (DIG) or soluble fractions were isolated from unstimulated or antiCD3/CD28-stimulated J.
1 and J.
1.WT-2 cells, and PKC
expression in different fractions was examined by immunoblotting. As shown previously (Bi et al., 2001; Villalba et al., 2001), stimulation induced PKC
translocation to the DIG-containing fractions (lipid rafts) in both cell lines (Fig. 1 D), albeit the distribution pattern of PKC
among the relevant fractions (24) differed between the two cell lines. Nevertheless, the overall amount of PKC
in fractions 24 was higher in J.
1.WT-2 cells when compared with the PLC
1-deficient J.
1 cells, suggesting some role for PLC
1. The same fractions were probed in parallel with a PLC
1-specific antibody. As expected, the J.
1 cells did not express detectable amounts of PLC
1 and, in agreement with previous results (Zhang et al., 2000), stimulation induced translocation of PLC
1 to the lipid rafts in the reconstituted (PLC
1 wt-2) cells (Fig. 1 E).
Activation of PKC enzymes is associated with their auto- or heterophosphorylation, events that regulate the enzymatic activity (Newton, 1997; Parekh et al., 2000). Although the regulation of PKC localization and/or activity by phosphorylation has not been analyzed in detail, a recent study indicated that an antibody specific for phosphorylated Thr-538 in the activation loop of PKC
reacted specifically with the active, membrane-localized fraction of PKC
(Bauer et al., 2001). We used another antibody specific for Ser-695 in the COOH-terminal tail of PKC
, which is a potential autophosphorylation (Keranen et al., 1995) or heterophosphorylation (Ziegler et al., 1999; Parekh et al., 2000) site based on its homology with other PKC enzymes in order to assess the role of PLC
1 in PKC phosphorylation. This site has very recently been implicated as a positive regulatory site in PKC
(Liu et al., 2002). As expected, this antibody did not recognize PKC
in unstimulated T cells, even though PKC
was readily detected by a PKC
-specific antibody (Fig. 2, two top panels). Anti-CD3 plus anti-CD28 stimulation induced the expected translocation of PKC
to the insoluble fraction, which represents the pooled membranes and cytoskeleton. Unlike the PKC
-specific antibody, the phospho-PKC
specific antibody only recognized PKC
from activated cells, which was exclusively associated with the insoluble fraction. Importantly, pretreatment of the cells with a selective PLC inhibitor (U73122, two middle panels) or its nonfunctional analog (U73343, two bottom panels) had no significant effect on the induction and membrane translocation of phospho-PKC
(Fig. 2).
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Defective membrane translocation of PKC in Vav-deficient primary T cells
Next, we decided to study the components of the unique pathway involved in the membrane translocation of PKC. First, we examined the role of Vav by comparing T cells from wild-type versus Vav-deficient T cells (Fig. 4). F-actin localization was determined in parallel. In order to expand the T cell population from the vav-/- mice, their lymph node cells were activated with an anti-CD3 mAb in the presence of IL-2, and then rested prior to restimulation. In T cells derived from vav+/+ mice, combined CD3/CD28 engagement induced actin polymerization, with a tendency of F-actin to polarize in a cap-like structure in a fraction of the cells. In agreement with previous results (Fischer et al., 1998; Holsinger et al., 1998), this outcome was clearly reduced in stimulated T cells derived from vav-/- mice (Fig. 4 A).
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The role of PI3-K in Vav-mediated membrane translocation of PKC
PI3-Kgenerated lipid products activate Vav and recruit it to the membrane by binding to its pleckstrin-homology (PH) domain (Han et al., 1998). Consistent with this finding, a PI3-K inhibitor blocked the membrane translocation of PKC in peripheral blood T cells (Fig. 1 B). Together, these findings suggest a role for PI3-K in activating the Vav pathway involved in PKC
membrane translocation. To address this potential role, we examined the effect of a transfected membrane-targeted (constitutively active) p110 plasmid or a PI3-K inhibitor on the membrane and cytoskeleton translocation of cotransfected PKC
in Jurkat-TAg cells. As a positive control, we cotransfected another group of cells with Vav, which induces PKC
translocation to these subcellular compartments (Villalba et al., 2000a).
In empty vector-transfected cells, anti-CD3 stimulation induced membrane translocation of PKC, which was reduced by LY294002 pretreatment (Fig. 5 A, top). Similar to Vav, p110 overexpression also induced PKC
translocation to the membrane as well as the cytoskeleton fractions in unstimulated cells, but no significant cooperation between Vav and p110 was observed; either Vav or p110 enhanced the ability of an anti-CD3 antibody to translocate PKC
(Fig. 5 B). Expression of p110, as well as anti-CD3 stimulation, also enhanced the membrane and cytoskeleton translocation of Vav (Fig. 5 A, two bottom panels). The PI3-K inhibitor LY294002 markedly inhibited both the p110- and Vav-induced PKC
translocation. However, it was less effective in Vav- plus p110-cotransfected cells, possibly reflecting the strong activating effect of this combined transfection and/or sufficient tyrosine kinase-mediated and PI3-K-independent Vav activation under these conditions.
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PDK1 is indirectly involved in the membrane translocation of PKC
PDK1 associates with, and is responsible for, activation loop phosphorylation of different PKC enzymes (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998; Balendran et al., 2000; Dutil and Newton, 2000; Gao et al., 2001). PDK1 and PKC need to be corecruited to the membrane through interaction with their respective membrane-localized allosteric activators in order for this phosphorylation to be efficient (Chou et al., 1998; Parekh et al., 2000; Toker and Newton, 2000; Sonnenburg et al., 2001). Calphostin C, which selectively blocks the allosteric activation of PKC by DAG, also inhibits serum-induced activation loop phosphorylation, as do PI3-K inhibitors (Parekh et al., 1999). These findings suggested an alternative mechanism for the membrane recruitment of PKC, i.e., its association with PDK1, which, by virtue of its PH domain, may localize PKC
to the membrane. Therefore, we examined the relative localization of PDK1 and PKC
in unstimulated or TCR-stimulated T cells.
When Jurkat T cells were stimulated with a combination of anti-CD3 plus -CD28 antibodies (or with PMA), endogenous PKC was clearly translocated to the membrane fraction; however, under the same conditions we could not detect similar translocation of endogenous PDK1 (Fig. 6 A), indicating that in T cells, PDK1 intracellular localization is not regulated by TCR/CD28 stimulation. To assess more directly whether PDK1 can influence the translocation of PKC
, we cotransfected Jurkat-TAg cells with PKC
plus PDK1 expression vectors. PDK1 coexpression enhanced the membrane and cytoskeleton translocation of PKC
, and this effect was only partially sensitive to a PI3-K inhibitor (Fig. 6, B and C). Interestingly, this enhanced PDK1-induced translocation of PKC
was largely reversed by coexpression of the dominant negative (
PH) Vav mutant. Even under these overexpression conditions, no PDK1 was detected in the membrane and cytoskeletal fractions of the stimulated cells (Fig. 6 C). In other, functional assays we found that coexpression of PDK1 with PKC
did not enhanced the PKC
-induced activation of NF-
B and AP-1 reporter genes (unpublished data). These results suggest that, although PDK1 may be involved in the maturation (perhaps via activation loop phosphorylation; Bauer et al., 2001) of PKC
in a similar manner to other PKC enzymes (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998; Dutil and Newton, 2000; Toker and Newton, 2000), it does not directly translocate PKC
to the membrane by associating with it in T cells.
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Discussion |
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In this study we sought to further define components of the selective pathway responsible for PKC membrane recruitment and, furthermore, the relative importance of the conventional PLC/DAG-mediated pathway in this event. First, we used three distinct approaches, i.e., a pharmacological PLC inhibitor, a PLC
1-deficient T cell line, and a dominant negative PLC
1 mutant to examine the role of PLC by comparing the behavior of PKC
to that of a representative T cellexpressed cPKC, PKC
. Each of these PLC-inhibiting strategies inhibited the membrane recruitment and/or activation of PKC
, but had, at best, a small effect on PKC
. In addition, we demonstrate that, like Vav (Villalba et al., 2000a), constitutively active PI3-K promotes membrane recruitment of PKC
, and that a PI3-K inhibitor blocks this event. Furthermore, we show that a dominant negative Vav mutant blocked PI3-K (p110)-induced PKC
translocation, suggesting that PI3-K functions either upstream of, or in parallel to, Vav to mediate PKC
membrane translocation. The finding that recruitment of PKC
to the cap formed by TCR/CD28 stimulation is largely absent in Vav-deficient T cells or is blocked by dominant negative Vav mutants in Vav-expressing T cells reaffirms the importance of Vav in this event.
The requirement for both Vav and PI3-K in PKC membrane translocation and activation most likely reflects the fact that Vav is a critical target for PI3-K in a single pathway regulating PKC
activation in the IS. This putative mechanism is consistent with the finding that Vav is activated by PIP3 binding to its PH domain (Han et al., 1998), a process that recruits Vav to the membrane and facilitates its catalytic activation by regulatory tyrosine phosphorylation (Aghazadeh et al., 2000). The requirement of TCR/CD28 costimulation for stable activation and membrane or lipid raft translocation of PKC
(Coudronniere et al., 2000; Bi et al., 2001) could reflect this dual regulatory mechanism for Vav activation. Thus, PI3-K is primarily stimulated by CD28 ligation (Rudd, 1996) and tyrosine kinases such as Lck and ZAP-70 are targets for TCR signals (van Leeuwen and Samelson, 1999; Kane et al., 2000). In this regard, CD28 has been shown to colocalize with PKC
at the IS (Monks et al., 1998), and lipid rafts, which accumulate at the IS in antigen-stimulated T cells (Bi et al., 2001), represent sites where PIP2, the precursor of PIP3, is formed in the membrane (Rozelle et al., 2000). Such a dual role for tyrosine kinases and PI3-K in Vav stimulation leading to PKC
activation is also consistent with our findings that PKC
membrane recruitment is inhibited by both Src family and PI3-K inhibitors. Finally, the finding that Vav and constitutively active PI3-K do not cooperate to enhance membrane translocation of PKC
(Fig. 5) is also consistent with the notion that PI3-K and Vav function in a single pathway. However, we cannot formally rule out the possibility that Vav and PI3-K function in two independent pathways to promote PKC
translocation and activation.
Although PDK1 overexpression induced prominent translocation of PKC to the membrane and to the cytoskeleton (Fig. 6), it itself did not undergo detectable membrane translocation upon T cell activation, even when overexpressed in T cells. These findings strongly suggest that direct association of PKC
with PDK1 does not occur in stimulated T cells and, therefore, most likely cannot account for the inducible membrane translocation of PKC
. Furthermore, if PDK1 associated with PKC
, PDK1 overexpression in the cytosol would be expected to retain PKC
in the cytosol and, thus, inhibit its anti-CD3induced translocation to the membrane, but we actually observed the opposite result. Thus, PDK1 may play an indirect role in the membrane translocation of PKC
, perhaps reflecting its ability to phosphorylate PKC
and induce its maturation, as demonstrated for other members of the PKC family (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998; Dutil and Newton, 2000; Toker and Newton, 2000). This effect appeared to be partially PI3-Kindependent, consistent with a recent report (Sonnenburg et al., 2001). However, the details of this indirect effect remain to be determined. At any rate, our finding that PDK1 does not increase PKC
-induced NF-
B or AP-1 activity (unpublished data) indicates that PDK1-mediated PKC
translocation is not sufficient to render it functional. Finally, the ability of dominant negative Vav to inhibit PDK1-induced PKC
translocation suggests that Vav may function downstream of PDK1. However, the two could function in separate pathways, a notion supported by the finding that, unlike Vav (Dienz et al., 2000; Hehner et al., 2000), PDK1 did not cooperate with PKC
to activate NF-
B (unpublished data).
Our results do not completely rule out a requirement for DAG binding to the PKC C1 domain in initiating its membrane binding and activation. It is possible that some residual level of basal DAG that remains even under conditions of blocked PLC activity is sufficient to initiate PKC
membrane binding. Albeit not sufficient for further recruitment of PKC
to specific membrane compartments such as the IS or lipid rafts, it may facilitate the interaction of PKC
with membrane or cytoskeletal component required for translocation of PKC
to the cSMAC and its full activation. Such a component could be some membrane-localized protein kinase that transphosphorylates PKC
or an adapter/scaffold protein that recruits it to specific membrane microdomains (Monks et al., 1997, 1998) or lipid rafts (Bi et al., 2001; Villalba et al., 2001). However, even if such a cooperative binding-activation mechanism exists, we still conclude that, unlike other PKCs, activated PLC and its lipid second messengers are not absolutely essential for PKC
IS translocation and activation.
In summary, our study defines a Vav-, PI3-K and, indirectly PDK1-dependent pathway(s), which selectively regulates the IS recruitment and activation of PKC in T cells. Thus, in addition to the conventional PLC/DAG-dependent pathway, the TCR/CD28 receptor system governs at least one additional pathway that positively regulates PKC function. Ongoing studies will define in more detail the molecular basis of this novel pathway.
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Materials and methods |
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Plasmids
The c-Myctagged Vav and VavPH expression plasmids in the pEF vector, an expression vector encoding the regulatory domain of PKC
fused to the NH2 terminus of GFP, Xpress-tagged PKC
, and the luciferase reporter gene plasmid driven by synthetic NFAT sites derived from the IL-2 promoter have been described (Villalba et al., 2000a). An HA-tagged, dominant negative PLC
1 mutant (PLCz) was a gift from Drs. Y. Abassi and K. Vuori (the Burnham Institute, San Diego, CA). This plasmid encodes the tandem SH2-SH2-SH3 domains of PLC
1 (Chen et al., 1996). A constitutively active PI3-K plasmid (CD2p110) in the form of membrane targeted PI3-K catalytic subunit (Reif et al., 1996) was provided by Dr. D. Cantrell (Imperial cancer Research Fund, London, England). A c-Myctagged PDK1 construct (Chou et al., 1998) was provided by Dr. Toshi Kawakami (La Jolla Institute for Allergy and Immunology, San Diego, CA). As control for transfection efficiencies, a ß-galactosidase (ß-gal) expression plasmid in the pEF vector was used (Villalba et al., 2000a).
Cell culture and transfection
Jurkat T cell lines were grown in RPMI-1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, MEM nonessential amino acid solution (Life Technologies) and 100 U/ml each of penicillin G and streptomycin. Cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as described (Villalba et al., 1999, 2000a). Human peripheral blood mononuclear cells were prepared from healthy volunteers by Ficoll-Hypaque centrifugation. Cells were stimulated with an activating anti-CD3 mAb (OKT3; 1 µg/ml) plus recombinant human IL-2 (20 U/ml) for 5 d, and then deprived of OKT3 and IL-2 36 h prior to restimulation. Mouse T cells were obtained from lymph nodes of Vav-/- or normal littermate mice, and purified on mouse T cell enrichment columns (R&D Systems). The cells were activated and rested as above, except an antimouse CD3 mAb (2C11-145; 1 µg/ml) was used.
Luciferase and ß-gal assays
Transfected Jurkat-TAg cells were harvested after 2 d, washed twice with PBS, and lysed. Luciferase or ß-gal activities in cell extracts were determined as described (Villalba et al., 1999). The results are expressed as arbitrary luciferase units per arbitrary ß-gal units. All experiments were performed in duplicate, and were repeated several times with similar results.
Subcellular fractionation
Subcellular fractionation of Jurkat T cells or peripheral blood mononuclear cells was performed as previously described (Villalba et al., 2000a). Briefly, Jurkat T cells were resuspended in ice-cold hypotonic lysis buffer, and incubated on ice for 15 min. The cells were transferred to a 1-ml syringe, and sheared by passing them five times through a 30-gauge needle. The lysates were centrifuged at 200 g for 10 min to remove nuclei and cell debris, the supernatant was collected, and centrifuged at 13,000 g for 60 min at 4°C. The supernatant (cytosol) was collected, and the pellet was resuspended in lysis buffer, vortexed for 5 min at 4°C, and centrifuged again at 13,000 g for 60 min at 4°C. The supernatant representing the particulate (membrane) fraction was saved, and the detergent-insoluble fraction (cytoskeleton) was resuspended in 1% SDS in water. Each fraction was then diluted to with Laemmli buffer, and identical cell equivalents separated by SDS-PAGE. The subcellular fractionation of activated human PBLs was similar. However, due to their small size, cells were incubated in hypotonic buffer lysis buffer in the presence of two drops of Polybead-polystyrene 4.5 micron microspheres (Polysciences, Inc.) with constant shaking in order to facilitate their disruption. In some experiments (Fig. 3), fractionation was not continued beyond isolation of the soluble (cytosol) and insoluble (membrane plus cytoskeleton) fractions in order to minimize dephosphorylation of PKC.
Purification of DIG fractions
Detergent-insoluble and soluble fractions were separated as described previously (Zhang et al., 1998; Bi et al., 2001) with some modifications. Briefly, Jurkat T cells (20 x 106) were lysed in 1 ml MNE buffer (25 mM MES, pH 6.5, 150 mM NaCl, 5 mM EDTA, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate and 10 µg/ml protease inhibitors) containing 1% Triton X-100 for 20 min on ice and dounced 15 times. Samples were centrifuged at 1,000 g for 10 min at 4°C. The supernatants were then mixed with 1 ml 80% sucrose and transferred to Beckman ultracentrifuge tubes. 2 ml of 30% sucrose followed by 1 ml of 5% sucrose in MNE buffer were overlaid. Samples were subjected to ultracentrifugation (200,000 g) for 18 h at 4°C in a Beckman SW50Ti rotor. 12 fractions were collected from the top of the gradient. Proteins from each fraction were TCA precipitated before separation by 10% SDS-PAGE.
Immunofluorescence and confocal microscopy
Jurkat cells were incubated with or without 1 µg/ml each of anti-CD3 and anti-CD28 mAbs for 10 min over poly-L-lysinetreated microscope slides at 37°C. Cells were then fixed for 20 min with 3.7% paraformaldehyde at room temperature, permeabilized for 2 min with 0.1% Triton X-100 in PBS, blocked for 15 min with 1% BSA in PBS, and then stained with phalloidin-TRITC (Sigma-Aldrich) for 30 min. After washing four times with 1% BSA in PBS, the cells were mounted using a drop of Aqua-Poly/mount (Polysciences). Samples were viewed with a Plan-Apochromat 63x lens on a Nikon microscope. Images were taken using BIORAD MRC 1024 laser scanning confocal imaging system.
Activated mouse T cells were similarly incubated over poly-L-lysinetreated microscope slides coated or not with 5 µg/ml of antimouse-CD3 plus-CD28 antibodies in Tris 50 mM, pH 9, for 1 h at 37°C, followed by 4 h at 4° C. Cells were then fixed and permeabilized as described above, and stained with a polyclonal anti-PKC antibody (C-18) for 1 h. The cells were washed with 1% BSA in PBS, and then incubated with a secondary sheep antimouse IgG antibody coupled with Alexa 594 (Molecular Probes) plus phalloidin-FITC. The cells were subsequently washed and processed for confocal microscopy as described above. Microsoft PowerPoint software was used to prepare digital images of gel scans and micrographs.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health Grants CA35299 and GM50819 (A. Altman). M. Villalba is a Special Fellow of the Leukemia & Lymphoma Society (formerly The Leukemia Society of America, Inc). This is publication number 426 from the La Jolla Institute for Allergy and Immunology, San Diego, CA.
Submitted: 22 January 2002
Accepted: 1 March 2002
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