Short Report 1357 αE- is not a significant regulator of β-catenin signaling in the developing mammalian brain

Wen-Hui Lien1,2, Olga Klezovitch1, Manda Null1 and Valeri Vasioukhin1,3,* 1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA 2Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98109, USA 3Department of Pathology and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA *Author for correspondence (e-mail: [email protected])

Accepted 31 January 2008 Journal of Cell Science 121, 1357-1362 Published by The Company of Biologists 2008 doi:10.1242/jcs.020537

Summary β-catenin is a crucial mediator of the canonical Wnt-signaling addition, we analyzed the expression of known endogenous pathway. α-catenin is a major β-catenin-binding , and targets of the β-catenin pathway and the amount and overexpressed α-catenin can negatively regulate β-catenin localization of β-catenin in mutant progenitor cells. We found activity. Thus, α-catenin may be an important modulator of the that although loss of αE-catenin resulted in disruption of Wnt pathway. We show here that endogenous α-catenin has intercellular adhesion and hyperplasia in the developing brain, little impact on the transcriptional activity of β-catenin in β-catenin signaling was not altered. We conclude that developing mammalian organisms. We analyzed β-catenin endogenous αE-catenin has no significant impact on β-catenin signaling in mice with conditional deletion of αE-catenin transcriptional activities in the developing mammalian brain. (Ctnna1) in the developing central nervous system. This mutation results in brain hyperplasia and we investigated whether activation of β-catenin signaling may be at least Supplementary material available online at partially responsible for this phenotype. To reveal potential http://jcs.biologists.org/cgi/content/full/121/9/1357/DC1 quantitative or spatial changes in β-catenin signaling, we used mice carrying a β-catenin-signaling reporter transgene. In Key words: Brain development, β-catenin signaling, α-catenin

Introduction β-catenin. Stabilized β-catenin associates with Lef/Tcf β-catenin is an (AJ) protein involved in both transcription factors and activates the transcription of multiple intercellular adhesion and regulation of the canonical Wnt including the classic targets of β-catenin pathway Myc, signaling pathway (Clevers, 2006). In adhesion, β-catenin links Ccnd1 (encoding cyclin D1) and Axin2 (He et al., 1998; Lustig et with α-catenin, and this interaction is crucial for the al., 2002; Tetsu and McCormick, 1999). Many β-catenin-binding assembly and maintenance of AJs (Perez-Moreno and Fuchs, play a profound role in the regulation of its signaling 2006). In Wnt signaling, β-catenin binds to the Lef/Tcf family of activities. Interaction of β-catenin with Lef/Tcf and BCL9/BCL9- transcriptional factors and functions as a transcriptional co- 2 is required for signaling (Behrens et al., 1996; Brembeck et al., activator (Clevers, 2006; Nelson and Nusse, 2004). Both the 2004; Kramps et al., 2002; Molenaar et al., 1996). By contrast, adhesion and signaling activities of β-catenin play a pivotal role in adenomatous polyposis coli (APC) and axin proteins are necessary normal development and tissue homeostasis and it is often difficult for degradation of β-catenin and inactivating mutations in Apc and to discern which of these functions is most critical at any given Axin2 result in abnormal activation of the Wnt pathway and time of development or in the adult life of an organism. In fact, predisposition to colorectal cancer (Behrens et al., 1998; Korinek such a central position in both intercellular adhesion and signaling et al., 1997; Morin et al., 1997; Polakis, 2007). makes β-catenin a critical node that may be involved in In addition to known downstream effectors of the classic Wnt orchestrating the behavior of individual cells assembled into a pathway, AJ proteins can also influence β-catenin transcriptional multicellular organism (Lien et al., 2006b). activity. For example, cadherins negatively regulate β-catenin by In addition to its role in normal development and adult tissue competing for interaction with Lef/Tcf factors and perhaps, by homeostasis, the β-catenin-signaling pathway also plays a causal tethering β-catenin to the cell plasma membrane away from the role in a variety of human malignancies (Clevers, 2006; Perez- nucleus (Cox et al., 1996; Fagotto et al., 1996; Heasman et al., Moreno and Fuchs, 2006; Polakis, 2007). Elucidation of the 1994; Orsulic et al., 1999; Sanson et al., 1996). It has been reported mechanisms responsible for regulation of β-catenin signaling is that another β-catenin-binding and AJ protein, α-catenin, is also a necessary for understanding of the Wnt pathway and developing negative regulator of β-catenin-signaling pathway (Giannini et al., efficient tools for anti-cancer therapy. The canonical Wnt signal 2000a; Hwang et al., 2005; Merdek et al., 2004; Sehgal et al., 1997; transduction pathway is the principal regulator of β-catenin- Simcha et al., 1998). Overexpression of α-catenin antagonized the mediated transcription (Clevers, 2006). Wnt interacts with its dorsalization effects of β-catenin in Xenopus embryos (Sehgal et receptor Frizzled, which activates a signaling cascade that al., 1997). In addition, overexpression of α-catenin in cell lines also ultimately results in attenuation of the activity of the β-catenin resulted in attenuation of β-catenin transcriptional activity destruction complex and accumulation of cytoplasmic and nuclear (Giannini et al., 2000b; Merdek et al., 2004; Simcha et al., 1998). 1358 Journal of Cell Science 121 (9)

Finally, siRNA-mediated depletion of α-catenin in cultured (Fig. 1A,B). Discrete protein bands corresponding to β-catenin, chondrocytes resulted in a small, but statistically significant cadherins, α-catenin and few additional unidentified proteins were increase in β-catenin signaling (Hwang et al., 2005). Negative present in the immunoprecipitates from wild-type brains (Fig. 1A). regulation of β-catenin transcriptional activity by α-catenin can be As expected, levels of α-catenin were significantly decreased in β- explained not only by sequestration of β-catenin away from the catenin protein complexes from αE-catenin–/– brains. Surprisingly, nucleus, but also by competition between α-catenin and DNA for this was the only major change that was detected by Coomassie binding to β-catenin/TCF complex (Giannini et al., 2000a). In Blue and Silver staining of the proteins immunoprecipitated with contrast to these studies, abnormal activation of β-catenin was not anti-β-catenin antibodies (Fig. 1A). We conclude that α-catenin is detected in mouse keratinocytes lacking the major epithelial α- a prominent part of β-catenin protein complexes; however, catenin, αE-catenin (Vasioukhin et al., 2001) and it is still unclear depletion of α-catenin has little effect on interaction between β- whether endogenous α-catenin can regulate the β-catenin-signaling catenin and other major β-catenin-binding proteins. Small amounts pathway in vivo. of α-catenin remaining in the complexes from mutant brains We have recently generated mice with conditional deletion of probably represent αN-catenin prominently expressed in the αE-catenin (Ctnna1) in the developing central nervous system and neurons of E14.5 brains (Fig. 1A), and the residual amounts of αE- found that these animals display massive brain hyperplasia and catenin in the few brain cells (endothelial cells) that remained dysplasia (Lien et al., 2006a). Although we revealed that abnormal untargeted by the -Cre transgene (Fig. 1B). activation of the Hedgehog-signaling pathway had an important To determine whether localization of β-catenin is changed in role in hyperplasia in αE-catenin–/– brains, an abnormal increase αE-catenin–/– brains, we performed immunofluorescent staining of in the total number of cells in the developing brain is also consistent cortical sections from E13.5 embryos with anti-N- and with the potential activation of β-catenin signaling. As multiple anti-β-catenin antibodies. Analysis of sections using confocal studies demonstrated that α-catenin is a negative regulator of β- microscope revealed disruption of apical junctional complexes and catenin transcriptional activity, we hypothesized that deletion of disorganization of αE-catenin–/– neural progenitors (Fig. 1C-D’’). αE-catenin in the developing mouse brain activates β-catenin and Nevertheless, β-catenin in these cells was still present at the cell that this activation may be at least partially responsible for periphery and colocalized with N-cadherin. We conclude that hyperplasia in αE-catenin–/– animals. Surprisingly, our results despite the depletion of αE-catenin, the composition of other major revealed no significant changes in the β-catenin-signaling pathway proteins in β-catenin protein complexes remains unchanged and in αE-catenin–/– mouse brains. These data suggest that endogenous that β-catenin continues to colocalize with N-cadherin at the α-catenin may have very little, if any, role in the regulation of β- surface of αE-catenin–/– neural progenitor cells. catenin transcriptional activity in vivo. Depletion of αE-catenin has no impact on the total level of Results and Discussion β-catenin and its nuclear localization αE-catenin is a prominent part of β-catenin protein complexes Wnt-mediated activation of β-catenin signaling usually results in Both αE-catenin and αN-catenin are expressed in the developing inhibition of the β-catenin destruction complex and accumulation mammalian brain; however, these genes display striking cell-type of β-catenin (Clevers, 2006). We analyzed potential changes in specificity, with αE-catenin primarily expressed in the dividing total levels of β-catenin in E12.5 and E13.5 wild-type and αE- neural progenitors and αN-catenin in differentiated neurons (Lien catenin–/– mouse brains using western blot analysis (Fig. 2A). et al., 2006a; Stocker and Chenn, 2006) (supplementary material Although αE-catenin was depleted in the mutant brains, the total Fig. S1). We previously reported that conditional deletion of αE- levels of β-catenin remained unchanged (Fig. 2A’). catenin in the developing central nervous system using a Nestin- Activated β-catenin localizes to the cell nucleus, which is Cre driver results in the disruption of cell-cell junctions, dysplasia indicative of activation of the β-catenin-signaling pathway. Nuclear and hyperplasia of neural progenitors (Lien et al., 2006a). Nestin- β-catenin is difficult to detect using regular immunofluorescent Cre is activated in neural progenitors at embryonic day (E) 10.5 of staining approaches. However, it can be revealed using special development (Graus-Porta et al., 2001). Although no differences in antigen-retrieval protocols (Merrill et al., 2001). We used such a cell numbers were detected between the wild-type and mutant protocol to reveal potential changes in the nuclear localization of brains in E12.5 embryos, E13.5 embryos displayed a 40% percent β-catenin in αE-catenin–/– mouse brains. No significant differences increase in the total number of cells in the mutant brains. The in the number or localization of cells with nuclear β-catenin were increase in cell numbers continued later in development; however, found between wild-type and αE-catenin–/– mouse brains (Fig. 2B- the most explosive hyperplasia in αE-catenin–/– brains took place C’’). We conclude that depletion of αE-catenin in the developing early, between days E12.5 and E14.5 (Lien et al., 2006a). Since mouse brain does not alter the overall level or nuclear localization αE-catenin is a known binding partner of β-catenin and β-catenin of β-catenin. plays an important role in regulating the proliferation of embryonic neural progenitor cells (Chenn and Walsh, 2002; Woodhead et al., An in vivo reporter for Lef/Tcf transcriptional activity reveals no 2006), we decided to analyze whether depletion of αE-catenin changes in the spatial distribution or level of β-catenin results in changes in transcriptional activity of β-catenin. Since the signaling in αE-catenin–/– brains most drastic hyperplasia took place in αE-catenin–/– brains from Although analysis of the total level of β-catenin or its nuclear E12.5 to E14.5, we concentrated on this developmental time point. localization can provide a general estimation of potential changes We first analyzed potential changes in β-catenin protein in the β-catenin signaling pathway, such measurements may not be complexes. For this purpose, β-catenin and its interacting proteins sufficiently sensitive and specific. The endogenous transcriptional were immunoprecipitated from wild-type and αE-catenin–/– brains reporter system has proved to be a very useful tool for quantitative and analyzed by SDS-PAGE followed by western blotting with and spatial analysis of β-catenin transcriptional activity. Several β- anti-β-catenin, anti-N-cadherin and anti-αE-catenin antibodies catenin-signaling reporter mice have been developed and analyzed Role of αE-catenin in β-catenin signaling 1359

Fig. 1. Depletion of αE-catenin has no major effect on interaction between β-catenin, N-cadherin and other β-catenin-binding proteins. (A,B) Total protein lysates from E14.5 wild-type (WT) and αE-catenin–/– (knockout, KO) mouse brains were immunoprecipitated with control (IgG) or anti-β-catenin (β-cat) antibodies and the resulting protein complexes were separated by SDS-PAGE and stained with Colloidal Blue and Silver stain (A) or analyzed by western blot with anti-αE-catenin, N-cadherin or β- catenin antibodies (B). Note that although αE-catenin becomes depleted from β-catenin protein complexes, composition or relative abundance of other proteins does not change. Western blotting reveals no significant changes in the association between β-catenin and N-cadherin. (C-D’’) Despite disruption of apical junctional complexes and loss of cell polarity, β-catenin continues to colocalize with N-cadherin at the periphery of αE-catenin–/– neural progenitor cells. Cortical sections from E13.5 wild-type (WT) and αE-catenin–/– (KO) embryos were stained with anti-N- cadherin (N-cad) and anti-β-catenin (β-cat) antibodies. Scale bar: 15.9 μm.

(DasGupta and Fuchs, 1999; Maretto et al., 2003; Moriyama et al., galactosidase antibodies. We found no significant differences in the 2007). To determine potential changes in β-catenin transcriptional level of the reporter between wild-type and mutant mouse brains activity in vivo, we used TOPGAL mice (DasGupta and Fuchs, (Fig. 3D). Overall, we conclude that neither spatial distribution nor 1999). These animals carry the transgene containing three Lef/Tcf the level of expression of the Lef/Tcf reporter construct were binding sites in front of a c-fos minimal promoter and LacZ reporter significantly altered in the αE-catenin–/– brain. (Fig. 3A) (DasGupta and Fuchs, 1999). We crossed our αE- cateninflox/flox/Nestin-Cre mice (Lien et al., 2006a) with TOPGAL Depletion of α-catenin has no impact on the levels of animals and generated αE-cateninflox/flox/Nestin-Cre/TOPGAL mice. endogenous transcriptional targets of β-catenin signaling Staining the brains of TOPGAL αE-catenin+/+ E12.5 and E13.5 The synthetic TOPGAL promoter may not be able to faithfully embryos for β-galactosidase activity revealed a pattern of reporter reproduce the complexity of transcriptional regulation at the expression that was similar to the pattern observed previously with endogenous promoter sequences, which are controlled by β-catenin other reporters of Lef/Tcf signaling (Maretto et al., 2003; Moriyama transcriptional activity. Although β-catenin can control et al., 2007). β-catenin signaling in the developing mouse transcription of many genes in a tissue- and time-specific manner, telencephalon was highly compartmentalized, with the reporter Myc, Ccnd1 and Axin2, are considered to be the classic endogenous displaying high levels of activity in the E12.5 dorsal cortex, transcriptional targets of β-catenin signaling (He et al., 1998; especially in the cingulate cortex area (Fig. 3B). In the E13.5 cortex, Lustig et al., 2002; Tetsu and McCormick, 1999). To examine the area displaying active β-catenin signaling expands to encompass whether depletion of αE-catenin results in changes in the the developing hippocampus (Fig. 3C). Remarkably, an almost transcriptional levels of these genes, we performed real-time PCR identical pattern of staining was observed in both E12.5 and E13.5 analysis using total RNA extracted from E12.5 and E13.5 wild- TOPGAL and αE-cateninflox/flox/Nestin-Cre/TOPGAL mice (Fig. type and αE-catenin–/– mouse brains. Although transition from 3B’-C’). Therefore, we conclude that depletion of αE-catenin does E12.5 to E13.5 is associated with significant hyperplasia in αE- not change the spatial pattern of active β-catenin signaling in the catenin–/– mice, we found no statistically significant changes in the developing mouse telencephalon. levels of Myc, Ccnd1 or Axin2 mRNA in wild-type and αE- Although staining of tissue sections for LacZ is useful for spatial catenin–/– mouse brains (Fig. 4). Thus, levels of the known localization of β-catenin signaling, quantification of this enzymatic endogenous transcriptional targets of β-catenin signaling are staining is challenging. To determine whether the overall levels of unchanged at the time of the most drastic increase in total cell the reporter were different in αE-catenin–/– brains, we performed number in αE-catenin–/– mouse brains. western blot analyses of the total protein extracts from TOPGAL In summary, we used a loss-of-function approach to determine wild-type and αE-catenin–/– mouse brains with anti-β- the potential role of endogenous αE-catenin in the regulation of β- 1360 Journal of Cell Science 121 (9)

Fig. 2. Depletion of αE-catenin has no effect on overall level of β-catenin or Fig. 3. Endogenous reporter for β-catenin transcriptional activity reveals no its nuclear localization. (A,A’) Total protein lysates from E12.5 and E13.5 changes in αE-catenin–/– mouse brains. (A) Schematic representation of wild-type (WT) and αE-catenin–/– (KO) mouse brains were analyzed by TOPGAL reporter (DasGupta and Fuchs, 1999). The reporter contains three western blotting with anti-αE-catenin, anti-αN-catenin, β-catenin and β- consensus Lef/Tcf-binding motifs (Lef) and a minimal c-fos promoter (P) to antibodies. Quantification of these results is shown in A’. Levels of β-catenin drive transcription of LacZ. (B-C’) A similar pattern of β-catenin reporter were normalized using β-actin and the results are shown as relative fold expression in the telencephalon of wild-type (WT) and αE-catenin–/– (KO) change. Data represent means ± s.d. (n=3). (B-C”) Sagittal telencephalon embryos. Sagittal sections of brains from E12.5 (B,B’) and E13.5 (C,C’) sections from E13 embryos were immunostained for nuclear β-catenin. embryos positive for TOPGAL transgene were stained for β-galactosidase Sections were subjected to antigen retrieval, stained overnight with anti-β- (blue) and counterstained with nuclear Fast Red. HI, developing hippocampus. catenin antibodies and processed using ABC MOM staining kit. Boxed areas Scale bar in B: 0.19 mm for B,B’ and 0.26 mm for C,C’. (D) Similar levels of in B and C are shown at higher magnification in B’,B” and C’,C”, respectively. β-catenin reporter expression were observed in wild-type (WT) and αE- β-catenin was present in the nuclei of progenitor cells, which were localized catenin–/– (KO) mouse brains. Total protein lysates from E12.5 and E13.5 around the ventricles. Prominent staining was also seen in the AJs at the telencephalons fromTOPGAL-positive animals were analyzed by western ventricular surface (black arrows). There were no significant differences in the blotting with anti-β-galactosidase (β-gal) and anti-β-actin antibodies. Con, nuclear β-catenin between the wild-type (WT) and knockout (KO) mouse control samples from TOPGAL-negative animals. The numbers indicate the brains. HI, developing hippocampus. Scale bar in B: 0.12 mm for B,C and relative amount of β-gal adjusted to the level of β-actin. 0.012 mm for B’-C’’.

catenin signaling in the developing brain. For this purpose, we loss of αE-catenin; however, this is unlikely, because αE-catenin–/– analyzed the level and localization of β-catenin, activity of β- progenitors display prominent cell-cell adhesion defects. Both αE- catenin signaling via a TOPGAL reporter construct and the levels catenin and αN-catenin are completely competent in AJ formation of known endogenous transcriptional targets of the β-catenin- (Hirano et al., 1992). Therefore, disruption of AJs in αE-catenin–/– signaling pathway. We did not find significant changes in β-catenin- progenitors indicates the absence of any compensation by αN- mediated transcriptional activity in αE-catenin–/– neural progenitor catenin. The most likely reason for differences between our results cells. Although it is possible that our analysis was not sensitive and previously published studies are the different model systems enough to detect potential minor changes in β-catenin signaling, our that were utilized. We believe that our in vivo approach may be results indicate that α-catenin has very little impact on β-catenin more relevant for the analysis of α-catenin, because the absence of signaling in vivo. This is different from the results obtained using a three-dimensional tissue organization in cells in culture may overexpression or knockdown of α-catenin in cultured cell lines introduce major changes, which are simply not pertinent to the (Giannini et al., 2000a; Hwang et al., 2005; Merdek et al., 2004; situation in the live organism. This is especially critical for studies Sehgal et al., 1997; Simcha et al., 1998). It is possible that small on , because these proteins are directly involved in tissue amounts of αN-catenin in neural progenitors compensate for the organization via their role in AJ formation. Role of αE-catenin in β-catenin signaling 1361

Fig. 4. Transition from E12.5 to E13.5 results in extensive hyperplasia in αE-catenin–/– brains without significant changes in the expression of endogenous transcriptional targets of the β- catenin-signaling pathway. (A) Hyperplasia in E13.5 αE-catenin–/– mouse brains. Total cells were isolated from E12.5 and E13.5 heterozygous and αE-catenin–/– mouse brains and counted using a Coulter Counter. Data represent means ± s.d. n=3-5. *P<0.0001. (B) qPCR analysis of β-catenin pathway transcripts Myc, Ccnd1 (cyclin D1) and Axin2 in E12.5 and E13.5 heterozygous and αE- catenin–/– mouse brains. The levels of expression are shown in arbitrary units with mean of levels in heterozygous embryos adjusted to 1. Data represent means ± s.d. n=4.

Materials and Methods RNA isolation and quantitative RT-PCR Mice Total brain RNA was extracted with Trizol and reverse-transcribed using SuperScript Mice with Nestin-Cre-mediated conditional deletion of αE-catenin in the developing III First-strand synthesis system kit (Invitrogen). Quantitative-PCR was performed central nervous system were generated as described (Lien et al., 2006a). TOPGAL using a Prism 7900HT instrument (Applied Biosystems), platinum qPCR mix Lef/Tcf reporter mice were obtained from Elaine Fuchs (DasGupta and Fuchs, 1999). (Invitrogen) and Universal ProbeLibrary kit utilizing the primers, probes and PCR To obtain TOPGAL/Nestin-Cre/αE-cateninflox/flox mice, we crossed TOPGAL conditions recommended by Universal ProbeLibrary assay center (www.roche- females with Nestin-Cre/αE-cateninflox/+ males, and the resulting TOPGAL/Nestin- applied-science.com/sis/rtpcr/upl/adc.jsp). PCR for ribosomal protein Rsp16 was Cre/αE-cateninflox/+ males were crossed with αE-cateninflox/flox females. All mice used for normalization. were on C57BL/6J genetic background. Total brain cell number counting Immunoprecipitation and western blotting To determine the total brain cell numbers, brains were dissected, incubated in DMEM μ Total proteins were extracted from embryonic brains with immunoprecipitation (IP) media containing 0.6 mg/ml papain (Worthington) and 20 g/ml DNase (Sigma) for buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Brij, 10% glycerol, 0.1 mM 20 minutes at room temperature, and dissociated to a single cell suspension by trituration. Cells were counted using Z1 Coulter particle counter (Beckman Coulter). EDTA, 0.5 mM MgCl2, 20 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate and a cocktail of protease inhibitors). Extracts were precleared by centrifugation at 25,000 g for 15 minutes at 4°C and supernatants were We thank all members of V. Vasioukhin’s laboratory for help and incubated with 30 μl of 50% slurry of Protein-A-Sepharose (Amersham) for 30 the critical reading of the manuscript; Akira Nagafuchi, Shoichino minutes. Resulting extracts were rotated at 4°C for 1 hour with anti-β-catenin Tsukita and the University of Iowa Developmental Studies Hybridoma μ antibody (Sigma) and then for 1 hour with 50 l of 50% slurry of Protein-A- Bank for generous gift of antibodies; Elaine Fuchs for the gift of Sepharose conjugated to rabbit anti-mouse antibody. Sepharose beads were washed four times with IP buffer and bound proteins were released by addition of LDS TOPGAL mice; and Vi Nguyen for help with mouse genotyping. This loading buffer and heating at 100°C for 5 minutes. Immunoprecipitated proteins or work was supported by NCI grant 1R01CA098161 to V.V. total protein extracts were resolved by NuPAGE electrophoresis (Invitrogen) and transferred to Immobilon P membrane (Millipore) or stained with Colloidal Blue or References α Silver stain (Invitrogen). The membranes were incubated with anti- E-catenin Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and (1:500, gift from Shoichino Tsukita, Kyoto University, Japan), anti-αN-catenin Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription (NCAT2, 1:500, University of Iowa Hybridoma Bank) anti-β-catenin (1:2000, factor LEF-1. Nature 382, 638-642. Sigma), anti-β-actin (1:10,000; Sigma), anti-N-cadherin (1:2000, Zymed), or anti-β- Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, galactosidase (1:2000, Rockland) antibodies at 4°C overnight. The membranes were M., Wedlich, D. and Birchmeier, W. (1998). 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