Article |
Address correspondence to Martin E. Hemler, Dana-Farber Cancer Institute, Rm. D1430, 44 Binney Street, Boston, MA 02115. Tel.: (617) 632-3410. Fax: (617) 632-2662. email: martin_hemler{at}dfci.harvard.edu
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Abstract |
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Key Words: integrins; tetraspanins; laminin-5; CD9; CD81
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Introduction |
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Tetraspanin proteins contain four transmembrane domains, one small and one large extracellular loop, and short, cytoplasmic NH2 and COOH termini (Berditchevski, 2001; Boucheix and Rubinstein, 2001). Tetraspanins may specifically regulate integrin-dependent cell motility and morphology, but typically do not affect static cell adhesion (Stipp and Hemler, 2000; Zhang et al., 2002). Tetraspanins associate with integrins, Ig superfamily proteins, membrane-bound growth factors, growth factor receptors, and other tetraspanins to form tetraspanin-enriched microdomains on the cell surface (Berditchevski, 2001; Boucheix and Rubinstein, 2001; Hemler, 2003).
To gain insight into tetraspanin function, we used mass spectrometry to identify novel associated proteins. Tetraspanins CD9 and CD81 were targeted because of their unique pattern of associated proteins (Stipp et al., 2001b), which includes EWI-2 (also called PGRL), as a member of a subfamily of four distinct but related IgSF proteins (Clark et al., 2001; Stipp et al., 2001a; Charrin et al., 2003a). In relatively stringent detergent conditions (1% Brij 96/97), EWI-2CD81 and EWI-2CD9 complexes are stable, fully soluble, limited in size (<4 million D), highly stoichiometric, and can be chemically cross-linked, indicative of direct proteinprotein interactions (Claas et al., 2001; Stipp et al., 2001a,b; Charrin et al., 2003a).
EWI-2 is widely expressed, with prominent mRNA expression in the brain (Clark et al., 2001; Stipp et al., 2001a), and protein expression on peripheral blood lymphocytes and hepatocytes, where it colocalizes with CD81 (Charrin et al., 2003a). We hypothesized that, as a major tetraspanin partner, EWI-2 might regulate cell motility on laminin, given the preferential association of tetraspanins with laminin-binding integrins. Our results establish EWI-2 as a novel regulator of cell reaggregation and motility on laminin-5 and reveal CD9 and CD81 as key linkers in a physical complex of EWI-2 with 3ß1 integrin, a laminin-5 receptor.
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Results |
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EWI-23ß1 complexes: a critical role for tetraspanins
Three lines of evidence indicate that tetraspanins play a key role in linking EWI-2 to 3ß1. First, tetraspanins CD9 and CD81 associate with both EWI-2 and
3ß1. Immunoprecipitations of CD9 (Fig. 7 B, lanes 46) and CD81 (Fig. 7 B, lanes 79) yielded both
3ß1 integrin and EWI-2, with higher levels of EWI-2 (Fig. 7 B, lanes 4 and 7) obtained from EWI-2overexpressing cells. Integrins (
2ß1,
6ß4) not associating with CD9 and CD81 at the same time failed to show associated EWI-2 (Fig. 7 B, lanes 1621). Second, removal of CD9 and CD81 resulted in diminished EWI-2
3ß1 association. After immunodepletion with anti-CD9 and -CD81 antibodies,
3 integrin immunoprecipitation yielded a level of EWI-2 (Fig. 8 A, lane 5) that was 85% reduced (as measured by densitometry) compared with that seen in control immunodepletions (Fig. 8 A, lanes 4 and 6). Removal of CD9 and CD81 was essentially complete, as seen in a subsequent CD81 immunoprecipitation (Fig. 8 A, lane 2), whereas mock immunodepletion or
2 integrin depletion failed to remove CD9 and CD81 or associated EWI-2 (Fig. 8 A, lanes 1 and 3). Conversely,
2 integrin was all removed by
2 immunodepletion (Fig. 8 A, lane 9), but not by mock or CD9/CD81 depletion (Fig. 8 A, lanes 7 and 8). Because CD9/CD81 depletion removed only a fraction (<10%) of total
3ß1 integrin (Fig. 8 A, compare lane 4 with lane 5), most
3ß1 appears not to be associated with CD9, CD81, or EWI-2. Third, the introduction of CD81 into CD81-deficient U937 monocytic leukemia cells (Hamaia et al., 2001) resulted in EWI-2
3ß1 association. Immunoprecipitation of
3 yielded FLAG-tagged EWI-2 when CD81 was present (Fig. 8 B, lane 6), but not when absent (Fig. 8 B, lane 5). Control CD81 immunoprecipitations from CD81+ cells yielded EWI-2 (Fig. 8 B, lane 4) and
3 integrin (Fig. 8 B, lane 8), as well as CD81 itself (Fig. 8 B, lane 12), whereas CD81 immunoprecipitations from CD81- cells did not (Fig. 8 B, lanes 3, 7, and 11). Conversely,
3 immunoprecipitation yielded CD81 only when CD81 was present (Fig. 8 B, compare lane 14 with lane 13). Immunoblotting of directly immunoprecipitated
3 confirmed that
3 levels were similar in CD81- and CD81+ cells (Fig. 8 B, lanes 9 and 10). Although smaller biosynthetic precursors of
3 were present in the total
3 blot (Fig. 8 B, lanes 9 and 10), exclusively the mature form of
3 integrin associated with CD81 (Fig. 8 B, lane 8), as previously observed (Kazarov et al., 2002).
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EWI-2 mutant (EW2xCD2c) retains 3ß1 association, but shows other biochemical deficiencies
Immunoprecipitation of FLAG-tagged EW2xCD2c and EWI-2 yielded similar amounts of associated 3 integrin as seen either by immunoblotting (Fig. 9 A, top; lanes 2 and 3) or by cell surface biotin labeling (Fig. 9 B, top; lanes 2 and 3). However, the EW2xCD2c protein showed substantially diminished association with CD81. CD81 was detected by blotting in immunoprecipitates of EWI-2 (Fig. 9 A, middle; lane 3), but not EW2xCD2c (Fig. 9 A, lane 2). Consistent with CD81-dependent maturation results in Fig. 8 B, the EW2xCD2c protein (with impaired CD81 association) showed a lower ratio of mature/immature forms (Fig. 9 A, bottom; compare lane 2 with lane 3). Consistent with retarded maturation, EW2xCD2c and EWI-2 showed similar levels of total FLAG-tagged protein (Fig. 9 A, bottom), but the amount of surface-labeled EW2xCD2c was somewhat diminished compared with EWI-2 (Fig. 9 B, compare lane 2 with lane 3).
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Evidence for extended EWI-2CD813ß1 complexes
Several experiments suggest that additional CD81 and EWI-2 molecules may be appended to EWI-2CD813ß1 and EWI-2CD9
3ß1 "core" complexes, but not to EW2xCD2c-CD9-
3ß1 core complexes. For example, immunoprecipitations of EWI-2 and EW2xCD2c yielded similar levels of associated
3 (Fig. 9, A and B), but immunoprecipitations of CD9, CD81, or
3 each yielded more EWI-2 than EW2xCD2c (Fig. 9 C, top; compare lanes 68 with lanes 24). In addition, from EWI-2transfected A431 cells, immunoprecipitations of EWI protein, CD9, or
3 each yielded more CD81 (Fig. 9 C, bottom; compare lanes 5, 6, and 8 with lanes 1, 2, and 4). In Fig. 9 D, immunoprecipitation of
3 again yielded much more CD81 from EWI-2expressing A431 cells (Fig. 9 D, lane 3), than from EW2xCD2expressing cells (Fig. 9 D, lane 2). Together, these results suggest that although EW2xCD2c is adequate for assembly into core (EWICD9
3ß1) complexes, its loss of CD81 association coupled with diminished surface expression limits the recruitment of additional CD81 and EWI proteins (Fig. 10; see Discussion).
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Discussion |
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EWI-2 neither associated with 2ß1 integrin nor affected reaggregation of A431 EWI-2 cells on collagen I, an
2ß1 ligand. Although EWI-2 did reduce the mean A431 cell migration rate on collagen I (by
3050%; unpublished data), this effect was not as great as that seen on laminin-5 (6070%). This result is consistent with
3ß1 functioning to some extent as a secondary receptor on a wide range of ligands to which it does not mediate initial attachment (DiPersio et al., 1995). The results also suggest a nonlinear correlation between cell migration and cell reaggregation, such that reaggregation is strongly affected by large changes in migration, but is disproportionately less affected by smaller changes in cell migration. Integrin
6ß4 also did not associate with EWI-2 and, compared with
3ß1, contributed much less to cell spreading on laminin-5. This is in accord with earlier analyses, in which
6ß4 integrin contributed to cell attachment on laminin-5, but
3ß1 was required for cell spreading (Xia et al., 1996; DiPersio et al., 1997).
Our results yield notable clues regarding the mechanism for EWI-2 effects on 3 integrindependent function. First, EWI-2 caused tetraspanin CD81 protein to localize into filopodia at the cell periphery, an ideal position in which to affect cell migration. Second, EWI-2 promoted an increased biochemical association of CD81 with
3ß1 integrin. A mutant form of EWI-2 (EW2xCD2c) that failed to associate with CD81, and also did not inhibit cell migration on laminin-5, in parallel failed to redirect CD81 to filopodia and failed to enhance CD81
3ß1 association. We propose that EWI-2dependent redirection of CD81 into proximity with filopodial
3ß1 should also lead to enhanced recruitment of CD81-associated cytoplasmic signaling enzymes, such as classical PKC isoforms and PI 4-kinase (Berditchevski et al., 1997; Zhang et al., 2001). These enzymes, together with other CD81-associated molecules, would then be in position to influence
3ß1-mediated cell migration.
Transmembrane proteins CD98, CD36, and CD47 all associate with various ß1 and/or ß3 integrins and modulate adhesive and/or motility functions (Thorne et al., 2000; Brown and Frazier, 2001; Fenczik et al., 2001). However, those molecules associate with integrins in the context of detergent-insoluble light membranes (Green et al., 1999; Thorne et al., 2000; Kolesnikova et al., 2001), whereas EWI-2tetraspanin and tetraspanin3ß1 associations occur within detergent-soluble fractions (Claas et al., 2001; Stipp et al., 2001a). Another member of the EWI subfamily, EWI-F/CD9-P1, can also associate with
3ß1 integrin (Charrin et al., 2003b), but the functional consequences have not yet been determined. In another analysis, EWI-2 overexpression impaired prostate cancer cell migration on laminin-1 and fibronectin (Zhang et al., 2003), but EWI-2 association with integrins was not studied, and the mechanism of EWI-2 function was not addressed.
EWI-2 association with 3ß1 is mediated by tetraspanins CD9 and CD81
Several observations point to EWI-2 association with 3ß1 being mediated by tetraspanins CD9 and CD81. First, in lysates depleted for CD9 and CD81, the majority of EWI-2 associated with
3ß1 was removed, even though the majority of
3ß1 itself remained undepleted. Thus, EWI-2 is linked, via tetraspanins, to a small but critical subset of
3ß1. Second, more integrin was recovered in a CD9 immunoprecipitation than in EWI-2 immunoprecipitation; and more EWI-2 was recovered by CD9 (or CD81) immunoprecipitation than by anti-
3ß1 (or CD151) immunoprecipitation. Again, this supports the idea that CD9 and CD81 are each more proximal to both the integrin and the EWI-2 than the integrin is to EWI-2. Third, CD9 and CD81 can associate with EWI-2 under conditions in which no integrin is present (Stipp et al., 2001a), and CD81 can associate with integrins under conditions in which no EWI-2 appears to be present (Zhang et al., 2001). Fourth, in U937 cells lacking CD9 and CD81, EWI-2 failed to associate with
3ß1 unless CD81 was coexpressed. Finally, the EW2xCD2 mutant, lacking CD81 association capability, instead used CD9 for linkage to
3ß1 (Fig. 10). These results strongly support a model in which either CD9 or CD81 can link EWI protein to
3ß1. Differences between CD9 and CD81, with respect to EW2xCD2 association, suggest that the EWI-2 cytoplasmic tail could be more important during CD81 association.
Our works reveal a novel function for CD81 in EWI-2 trafficking. First, in the absence of CD81 (or CD9), little EWI-2 underwent full biosynthetic maturation and appeared on the surface of U937 cells. Second, the EW2xCD2c mutant, deficient in CD81 association, also showed impaired maturation and cell surface expression. In the latter case, retention of CD9 association likely accounted for a significant fraction of EW2xCD2c maturing, reaching the cell surface, and engaging 3ß1 to the same extent that EWI-2 engages
3ß1. The finding that only
10% of the
3ß1 is involved in a complex with tetraspanins and EWI-2 helps to explain why EW2xCD2c and EWI-2 both engage a similar fraction of
3ß1, despite the lower cell surface expression of EW2xCD2c. The 10%
3ß1 estimation is consistent with previous estimates of
3ß1tetraspanin stoichiometry (Berditchevski et al., 1996). However, for the critical subset of
3ß1 molecules engaged with ligand in filopodia at the cell periphery, EWI-2tetraspanin
3ß1 association may be much greater than 10%.
Expanded EWI-2CD813ß1 complexes include additional CD81 and EWI-2 molecules
Both EWI-2 and the EW2xCD2c mutant were similarly linked via tetraspanins (CD81 and/or CD9) to the 3ß1 integrin. However, EWI-2 suppressed cell migration and enhanced the recruitment of CD81 to critical filopodial sites, whereas the EW2xCD2c mutant had neither of these activities. To explain these key functional differences, we look beyond the core EWItetraspanin
3ß1 complexes and consider the role of expanded complex formation. As illustrated in Fig. 10, both mutant and wild-type EWI-2 are linked to a similar extent to
3ß1, with EW2xCD2c using CD9, and EWI-2 using CD9 or CD81. However, with CD81 association leading to more EWI-2 cell surface expression, there is capability to assemble additional CD81 and EWI-2 molecules into an expanded complex. Hence, CD81 not only links EWI-2 to
3ß1, but EWI-2 also helps to recruit more CD81 to
3ß1. The tendency of CD81 and CD9 to exist as homodimers (Kovalenko et al., 2003) should facilitate extension of the complex. Lacking an ability to associate with CD81, the EW2xCD2c mutant cannot support expanded complex formation.
Tetraspanin-enriched microdomains contain cholesterol, gangliosides, tetraspanins, and other proteins. These microdomains, held together at least partly due to tetraspanin palmitoylation, are physically and functionally distinct from lipid rafts (Berditchevski, 2001; Boucheix and Rubinstein, 2001; Hemler, 2003). Our EWI-2tetraspanin3ß1 results extend the "tetraspanin microdomain" idea by demonstrating that multiple nontetraspanin proteins (an integrin and EWI-2) can be physically and functionally linked through specific tetraspanin proteins. The association of EWI-F/CD9-P1 with
3ß1 integrin provides another potential example of tetraspanins linking two nontetraspanin proteins, although the tetraspanins involved were not formally defined (Charrin et al., 2003b). Other examples of multiprotein complexes potentially linked by tetraspanins, such as
4ß1 and
6ß1 integrin with HLA-DR, (Rubinstein et al., 1996) and HB-EGF with ß1 integrin (Nakamura et al., 1995), relied on mild CHAPS detergent conditions in which tetraspanin complexes can have sizes in excess of 20 million D (Skubitz et al., 2000). Also, the linker function of tetraspanins was not directly tested in these experiments, and the functional relevance of the complexes was not established. Complexes of CD19 with CD21 (Horvath et al., 1998), and CD36 with
3ß1 or
6ß1 integrin (Thorne et al., 2000) are stable in Brij96 and may be functionally relevant, but the role of tetraspanins as linkers in these complexes remains untested. In another work, we found that EWI-2 can interact with another CD81-associated integrin (
4ß1) in the MOLT-4 T cell line to cause formation of expanded EWI-2CD81
4ß1 complexes, leading to alterations in
4ß1-dependent cell morphology (unpublished data).
Possible relevance in vivo
In zones of activated basement membrane underlying injury sites, cell migration on laminin-5 is mediated primarily by 3ß1 integrin (Nguyen et al., 2000). Laminin-5 can also trigger
3ß1-dependent scattering of squamous carcinoma cells (Kawano et al., 2001), suggesting a potential role for
3ß1 in metastasis. On the other hand,
3ß1 overexpression in the suprabasal layer of the epidermis inhibited malignant conversion of papillomas (Owens and Watt, 2001), suggesting a potential role for
3ß1 in suppressing tumor progression in some circumstances. In all of these cases, EWI-2 could play a key regulatory role, given its relatively widespread distribution (Clark et al., 2001; Stipp et al., 2001a). In addition, EWI-2 could participate in several key developmental processes. The defects of
3-null mice in kidney, lung, and skin morphogenesis point to a critical role for
3ß1 in the assembly or organization of laminin-5 in the basement membranes of these tissues (Kreidberg et al., 1996; DiPersio et al., 1997). EWI-2 may participate in these functions by regulating the outcome of
3ß1 engagement of laminin-5.
In conclusion, we have established a novel structural and functional link, via CD9 and CD81, between EWI-2 and the 3ß1 integrin. These results contribute to the emerging importance of tetraspanin-containing protein microdomains, and also establish a novel means of regulating integrin function through lateral interactions among transmembrane proteins.
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Materials and methods |
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Cell culture and retroviral transduction
A431 epithelial carcinoma cells and retroviral packaging cell lines NX and PT67 were cultured in high glucose DME, and U937 pro-monocytic leukemia cells in RPMI 1640. All cultures were supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all purchased from GIBCO BRL). Expression constructs were (1) a COOH-terminally FLAG-tagged EWI-2 in the pLXIZ retroviral vector (Stipp et al., 2001a); (2) also in pLXIZ, an EWI-2/CD2 chimera (EW2xCD2c) with the EWI-2 cytoplasmic tail (from Cys604) replaced by recombinant PCR with the tail of CD2 followed by the FLAG epitope; (3) human CD81 in the pLXIN retroviral vector; (4) human
3 integrin in pLXIZ; and (5) the empty pLXIZ vector. Expression vectors were transfected by calcium phosphate into
NX packaging cells. 48 h after transfection,
NX cell supernatants were passed through a 0.45-µm filter, supplemented with 4 µg/ml Polybrene® (Sigma-Aldrich), and used to infect PT67 packaging cells that had been pretreated with 200 ng/ml tunicamycin (Sigma-Aldrich) for 18 h. Stable PT67 packaging cells were obtained by selection for 2 wk in 500 µg/ml ZeocinTM (Invitrogen) or G418. The supernatant from these PT67 cells was harvested, filtered, and supplemented with Polybrene® as above, and then used to infect A431 cells or U937 cells. Stable infectants were selected and grown as uncloned populations.
Cell spreading assay
Serum-free medium (SFM) contained DME with 5 mg/ml cell culture grade BSA (#194771; ICN Biomedicals) and 20 mM Hepes, pH 7.2. A431 EWI-2 cells and A431 IZ cells were rinsed, detached by trypsin/EDTA treatment, and collected in SFM plus 0.1 mg/ml soybean trypsin inhibitor and 0.1 mg/ml DNase I (both purchased from Worthington Biochemical Corp.). After centrifugation, the supernatant was removed and cells were resuspended in SFM alone or SFM with (1) 5 µg/ml GoH3 anti-6, (2) 10 µg/ml A3-IIF5 anti-
3, or (3) both antibodies. Cells were then plated on acid-washed glass coverslips that had been coated with 1 µg/ml rat laminin-5 (provided by Desmos, Inc.) and blocked with SFM. Cells attached and spread for 20 min before being fixed with 3.7% formaldehyde in PBS with 5% sucrose and 2 mM MgCl2 for 15 min at RT. Coverslips were rinsed and mounted on slides in FluoroSave reagent (Calbiochem). Cell images were acquired with a monochrome CCD camera (Spot RT; Diagnostic Instruments) on a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.), and were controlled by IP Lab software (Scanalytics) running on a G4 Macintosh computer. Areas of cells (2045 cells per coverslip) were calculated using the Scion Image v1.62 program (Scion Corp.).
Reaggregation assay
Acid-washed glass coverslips (12-mm circles) were coated overnight at 4°C with 0.5 µg/ml rat laminin-5 in PBS with 0.005% Tween 20, or with 40 µg/ml collagen I in PBS. Coverslips were then blocked for 1 h at RT with SFM. A431 EWI-2 and A431 IZ cells that had been starved overnight in SFM were harvested as for the spreading assay above, and 1.5 x 105 cells per well were plated to laminin-5 or collagen Icoated coverslips in a 24-well dish. After 56 h, cells were fixed, stained with DAPI HCl (Sigma-Aldrich) to label nuclei, mounted, and imaged as above. Clustered and unclustered cells were counted in five fields per coverslip (typically >500 cells per coverslip). Two coverslips per condition were examined in each experiment, and always yielded statistically similar results. DAPI staining enabled unambiguous scoring of cell numbers in large, tightly packed cell clusters. Viability of A431 EWI-2 cells and A431 IZ cells under these assay conditions was tested using thiazolyl blue tetrazolium bromide (MTT) in Cell Proliferation Kit I (Roche).
Immunofluorescent localization
A431 cells were plated on laminin-5 in SFM. After 3 h, cells were fixed for 15 min with prewarmed 4% PFA, 2 mM MgCl2, and 4% sucrose in PBS. Fixed cells were rinsed three times with 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl, and were blocked for 30 min in 20% goat serum in PBS. Primary antibodies were added at 5 µg/ml in blocking buffer and allowed to bind overnight at 4°C. After rinsing in PBS, FITC-goat antimouse second antibody was added in blocking buffer for 1 h at RT. Rinsed coverslips were mounted in FluoroSave (Calbiochem). Fluorescent images were acquired on an upright microscope (Axioskop; Carl Zeiss MicroImaging, Inc.) using a Spot RT camera driven by IP Lab software, as described above.
Immunoprecipitation, immunoblotting, and immunodepletion
A431 cells were lysed by scraping into 20 mM Hepes, pH 7.5, 150 mM NaCl, and 5 mM MgCl2 (HBSM) supplemented with 1% Brij 96 (Fluka), 2 mM PMSF (Sigma-Aldrich), 20 µg/ml aprotinin, and 10 µg/ml leupeptin (Boehringer). In some experiments, cells were biotinylated with 0.2 mg/ml sulfo-NHS-LC biotin (Pierce Chemical Co.) in HBSM for 1 h at RT, then rinsed three times with HBSM before lysis. After a 1-h extraction at 4°C with rocking, insoluble material was removed by centrifugation, and lysates were precleared for 1 h at 4°C with protein GSepharose (BD Biosciences). M2 anti-FLAG agarose or specific antibodies plus protein GSepharose were added, and immune complexes were collected overnight at 4°C. After rinsing four times with lysis buffer, immune complexes were eluted by boiling in sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose. Blots were blocked with 5% nonfat milk in PBS with 0.1% Tween 20.
FLAG epitope immunoblots were rinsed with TBS and blotted for 30 min with biotinylated M2 anti-FLAG mAb (10 µg/ml in TBS). After 10 rinses, blots were developed with a 30-min exposure to HRP-ExtrAvidin® in TBS, followed by 10 more rinses, and chemiluminescence detection (Renaissance® reagent; NEN Life Science Products). Myc epitope and CD151 immunoblots were developed for 1 h with anti-myc 9E10 ascites or the 8C3 anti-CD151 mAb diluted in blocking buffer, followed by 1 h with an HRP-goat antimouse pAb and chemiluminescence.
For immunodepletion, cell surfacebiotinylated cells were lysed in 1% Brij 96 as described above. The lysate was divided into four parts and precleared three times for 1 h with (1) protein GSepharose alone, (2) protein GSepharose plus a combination of M38 and ALB6, anti-CD81, and CD9 antibodies, or (3) protein GSepharose plus A2-IIE10 anti-2 integrin. The depleted samples were further divided into three parts each, immunoprecipitated with agarose-conjugated anti-CD81, anti-
3, or anti-
2 antibodies, and then analyzed by SDS-PAGE and chemiluminescence as above. Semi-quantitative densitometry was performed using GeneTools software (Syngene) on digitized images captured from trans-illuminated films with a CCD camera driven by GeneSnap software (Syngene).
Online supplemental material
Video 1 shows that EWI-2 overexpression impairs carcinoma cell motility on laminin-5. A431 IZ cells (left) and A431 EWI-2 cells (right) were plated on laminin-5 in serum-free medium. Migration was monitored for 2 h at 1 frame/min, and the video play rate is 6 frames/s. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200309113/DC1.
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Acknowledgments |
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Submitted: 18 September 2003
Accepted: 14 October 2003
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References |
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