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1 Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
2 PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama, Japan
3 Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
4 Genetic Strains Research Center, National Institute of Genetics, 1111 Mishima, Shizuoka 411-8540, Japan
5 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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-Secretase (Brou et al. 2000; Struhl & Greenwald 2001). The intracellular domain of N is then liberated from the plasma membrane and translocates to the nucleus, where it acts as a co-activator of transcription (Schroeter et al. 1998; Struhl & Adachi 1998). N is expressed in various epithelial tissues, such as the Drosophila neuroectoderm and imaginal disc, during cell-fate specifications, in which N signaling plays crucial roles (Fehon et al. 1991). In these apicobasally polarized epithelial cells, the plasma membranes are subdivided into apical and basolateral domains that are separated by specialized junctional structures, the adherens junction (AJ) and septate junction (reviewed by Müller 2000; Tepass et al. 2001; Roh & Margolis 2003). The formation and stabilization of these junctions are paramount for the maintenance of epithelial polarity, which, in turn, is required for proper epithelial cell physiology, cell motility, asymmetric division, and intercellular signaling (reviewed by Tepass et al. 2001).
Many of these processes rely on the polarized distribution of plasma membrane proteins. Recent studies revealed that the polarized organization of post-Golgi trafficking, which depends on actin and microtubules, is important for the guidance of apical and basolateral proteins (reviewed by Rodriguez-Boulan et al. 2005). In addition, some sorting signals, such as N-glycans and O-glycans, and proteins for the docking and fusion of vesicles in polarized trafficking have been identified (reviewed by Rodriguez-Boulan et al. 2005).
N and Dl are localized to the apical region of epithelial cells in Drosophila (Fehon et al. 1991; Bender et al. 1993; Kooh et al. 1993). Hence, the N signaling pathway must be integrated into the concept of how these polarized structures in epithelial cells function. However, the mechanisms and function of N's apical localization are unknown. Here, we investigated how N and its ligands are delivered to specific regions of the polarized epithelial cells in the third-instar larval wing disc. We suggest that the apicobasally polarized structure may be a general requirement for the juxtacrine cell signaling that takes place in epithelial cells.
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Results |
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In the apical region of epithelial cells where N is located, we first investigated by studying the third-instar larval wing disc. To study the Notch (N) localization at high resolution, we performed deconvolution analysis of confocal microscope images. The wing disc epithelium develops typical apicobasally polarized structures (Woods et al. 1997; Müller 2000). As shown schematically in Fig. 1A, the SAC, AJs, and lateral region were distinguished by antibody staining with anti-atypical protein kinase C (aPKC), anti-DEcad, and anti-Discs large (Dlg) antibodies, respectively. These antibodies specifically defined these three regions, because the staining patterns of the anti-DEcad and anti-aPKC, and with anti-DEcad and anti-Dlg, did not overlap (Supplementary Fig. S2A,B). The localization of aPKC and N overlapped, indicating that most of the N was localized to the SAC (Fig. 1B). N staining overlapped with the apical portion of DEcad and of Armadillo, which labels AJs (Fig. 1C and data not shown). In contrast, N and Dlg, which marks septate junctions, were detected in mutually exclusive patterns (Fig. 1D). Therefore, N associated with the plasma membrane was concentrated at the SAC and AJs (SAC/AJs) in the wing disc epithelium. We also found that Dl and Ser were predominantly localized to the SAC/AJs and colocalized with N in these cells (Supplementary Fig. S1A–D). N localized normally to the SAC/AJs in cells that were double mutants for Dl and Ser (Fig. 1E). In addition, the ligands’ localization was unaffected in cells bearing a N mutation (Fig. 1F). Thus, N and its ligands localized to the SAC/AJs independently.
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The EGF domains of N receptors are modified by the addition of fucose to serine or threonine residues (O-fucose) (Moloney et al. 2000). This O-linked fucosylation is catalyzed by the GDP-fucose protein O-fucosyltransferase (O-FucT-1), O-fut1 in Drosophila (Wang et al. 2001), and this fucosylation is essential for N signaling and its ligand interactions (Lei et al. 2003; Okajima & Irvine 2002; Okajima et al. 2003; Sasamura et al. 2003). N did not localize normally to the SAC/AJs in somatic clones of an O-fut1 mutant (O-fut1–) (Fig. 1G). However, the localization of Dl to the SAC/AJs was not affected in the O-fut1– cells (Fig. 1H). Therefore, although both N and Dl localized to the SAC/AJs, their requirement for O-fut1 was different. O-fut1 is reported to act as a chaperon for N, independent of its O-fucosyltransferase activity, and misfolded N accumulates in the endoplasmic reticulum (ER), owing to the quality-control mechanism (Okajima et al. 2005). Thus, it is possible that N failed to localize to the SAC/AJs because of misfolding or defective transportation to the plasma membrane due to the lack of the enzymatic activity-independent function of O-fut1. Alternatively, a lack of N O-fucosylation could be responsible for the failure of N localization.
To distinguish among these possibilities, we studied the distribution of N in mutants of the GDP-D-mannose 4, 6-dehydratase (GMD) gene. In Drosophila, GDP-fucose is thought to be synthesized only through the de novo pathway, for which GMD is indispensable (Roos et al. 2002). Homozygotes of GmdH78, which lack most Gmd exons, barely survived to the third-instar larval stage (data not shown; Okajima et al. 2005), and N failed to localize to the SAC/AJs in the epithelial cells of their wing discs, suggesting that the O-fucosylation of N is essential for its localization to the SAC/AJs (Fig. 1I). In GmdH78 wing discs, N accumulation in the intracellular vesicles, which occurred in the O-fut1– cells, was not observed (Fig. 1I). This was most likely because the N accumulation is caused by the lack of an O-fut1 function that is independent of its O-fucosylation enzymatic activity, as proposed previously (Okajima et al. 2005), and this activity is maintained normally in Gmd mutants. In addition, we found that, in the cells of the GmdH78 wing discs, N was expressed in vesicles of endocytic origin (T.S., I.H.O., N.S., Syunsuke Higashi, M.K., Shiho Nakao, Tomonori Ayukawa, Toshiro Aigaki, Katsuhisa Noda, Eiji Miyoshi, Naoyuki Taniguchi, & K.M, submitted). Therefore, the O-fucosylation of N is not an essential requirement for its endocytic transportation. On the other hand, both O-fut1 and Gmd are indispensable for the normal localization of N to the SAC/AJs, suggesting that the O-fucosylation of N is essential for this trafficking process. Conversely, Dl was still localized to the SAC/AJs in these mutant discs (Fig. 1J), although it is a known substrate for O-fut1 (Panin et al. 2002). Therefore, O-fucosylation of N is specifically required for its proper localization in epithelial cells.
Delivery of nascent N by polarized exocytosis did not require O-fut1
We next investigated how N localizes to the SAC/AJs. First, we examined whether N was targeted to these structures during exocytosis. To address this issue, we expressed N+-GV3 under the control of a heat-shock (HS) promoter in the epithelial cells of the third-instar larval wing disc. N+-GV3 is a N derivative with a Gal4-VP16 domain insertion just below the transmembrane domain, and is otherwise wild-type (Struhl & Adachi 1998). N+-GV3 functions like wild-type N in vivo (Struhl & Adachi 1998). The subcellular localization of N+-GV3 was detected with an anti-Gal4 antibody at various time points. We also investigated how the delivery of nascent N was affected in O-fut1– cells, in which N fails to localize to the SAC/AJs, which we hoped would give us insight into the mechanisms of this N localization.
The expression of N+-GV3 was not detected either before HS or 0 min afterwards in wild-type or O-fut1– cells (Fig. 2A,B). N+-GV3 was first observed as small vesicles in the basal region of wild-type and O-fut1– epithelial cells 5 min after HS (Fig. 2C, arrowheads in vertical section). Large numbers of vesicles containing N+-GV3 were observed at the level of the AJs 15–30 min after HS. Some of these vesicles colocalized with DEcad in wild-type and O-fut1– cells (Fig. 2D,E). These data suggested that nascent N is delivered to AJs or their vicinity by exocytotic vesicles. Furthermore, we found that the majority of these vesicles did not colocalize with ER (protein disulfide isomerase (PDI)-GFP), Golgi (GM-130), or early endosome (Hrs) markers in wild-type and O-fut1– cells (Fig. 2F–H), consistent with the idea that N+-GV3 was in post-Golgi transport vesicles under these conditions (Lloyd et al. 2002; Bobinnec et al. 2003; Yano et al. 2005).
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Nascent N accumulated at SAC/AJs via a transcytotic pathway
We had observed the first difference in N localization in O-fut1– cells compared with wild-type cells at 45 min after HS. Thus, the appropriate localization of N to the SAC/AJs seemed to depend on some O-fut1 function at this time point. Vesicles containing N+-GV3 at the level of the AJs became larger 45 min after HS in both wild-type and O-fut1– cells. In wild-type cells, 48% of these large vesicles containing N+-GV3 colocalized with the AJs (n = 588) (Fig. 4A,C). In contrast, in the O-fut1– cells, only 13% of these vesicles colocalized with the AJs (n = 223) (Fig. 4A,C). N+-GV3 localization overlapped with the SAC/AJs in a "honey-comb" pattern in wild-type cells, but not in O-fut1– cells, 90 min after HS (Fig. 4B). Thus, there was a characteristic time lag between the delivery of nascent N by post-Golgi transport vesicles and the accumulation of N at the AJs.
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Next, we examined whether endocytosis is required for the localization of N to the SAC/AJs. It was demonstrated that the disruption of Dynamin function efficiently suppresses transcytosis in Drosophila epithelial cells (Parks et al. 2000). Dynamin is required during endocytosis for the formation and pinching off of clathrin-coated vesicles from the plasma membrane (reviewed in Le Borgne et al. 2005). shibireTS1 (shiTS1) is a temperature-sensitive allele of the Drosophila Dynamin gene (Grigliatti et al. 1973; van der Bliek & Meyerowitz 1991). A reduction in shi function in the developing wing disc at the non-permissive temperature (32 °C) for 20 min resulted in a decrease in the amount of N at SAC/AJs (Fig. 5B compared with 5A). Because a reduction in Dynamin function for only a short time was sufficient to disrupt the localization of N to SAC/AJs, it is likely that inhibition of Dynamin-dependent transcytosis directly affected the localization of N to SAC/AJs. The localization of N to the SAC/AJs was drastically disrupted in shiTS1 wing discs at 32 °C after 8 h (Fig. 5C). In these wing discs, N was mostly detected in vesicles in the apical region (Fig. 5C, vertical section). In contrast, the localization of DEcad was largely unaffected by holding the discs at 32 °C for 20 min or for 8 h (Fig. 5B,C). Under these conditions, ectopic apoptosis was not induced (data not shown). Furthermore, the localization of N and DEcad in wild-type discs was not affected after 20 min or 8 h at 32 °C (Fig. 5D and data not shown). These results suggest that the Dynamin-dependent transcytosis of N is required for its localization to the SAC/AJs.
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The localization of N and Dl to the SAC/AJs required DEcad
Disruption of the AJs is known to prevent proper SAC formation (Bilder et al. 2003; Tanentzapf & Tepass 2003). The expression of double-stranded RNA (dsRNA) corresponding to DEcad mRNA, driven by patched-Gal4 (ptc-Gal4) or MS1096-Gal4, reduced the level of DEcad protein (Fig. 7B, magenta and data not shown). However, the knock down of DEcad also induced apoptosis under these conditions (data not shown). Thus, to suppress this apoptosis, we simultaneously expressed DIAP (Inhibitor of apoptosis 1), which effectively blocked the apoptosis, with the dsRNA of DEcad (Kuranaga et al. 2002). We found that N and Dl failed to localize to the SAC/AJs in these cells (Fig. 6A). This result was not owing to a reduced amount of N and Dl (Fig. 6B). The knock down of DEcad in the wing discs disrupted the localization of aPKC, a marker for the SAC, while basolateral components, such as septate junctions, marked by Cora, still formed, suggesting the epithelial structure was maintained, at least in part (Fig. 6C). In addition, N did not colocalize with the vast majority of the aPKC in DEcad knock down cells, although N was still localized to the apical region of these cells (Fig. 6D).
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DEcad was required for epithelial N signaling
Given that the localization of N to SAC/AJs required DEcad, N signaling might depend on DEcad expression on polarized epithelial cells. Wingless (Wg) and Cut are targets of N signaling expressed in the dorsoventral compartment boundary of the third-instar larval wing disc (Fig. 7A,C) (Neumann & Cohen 1996). We found that the knock down of DEcad by RNAi reduced the Wg and Cut expression, when cell death was prevented by DIAP expression, indicating that N signaling was suppressed (Fig. 7B, arrowhead and data not shown). In contrast, as indicated by the arrows in Fig. 7B, Wg expression around the wing pouch was expanded. Fat signaling is known to inhibit this Wg expression in wild-type cells (Cho & Irvine 2004). The fat gene encodes a transmembrane receptor that is activated by the transmembrane ligand, Dachsous (Mahoney et al. 1991; Clark et al. 1995). This expansion in Wg expression suggested that Fat signaling decreased in these cells (Cho & Irvine 2004). These results suggest that the knock down of DEcad generally interferes with cell–cell interactions that are mediated by interactions between transmembrane receptors and ligands.
On the other hand, over-expressed DEcad driven by MS1096-Gal4 in the wing pouch (Fig. 7F) increased the number of cells expressing Cut at 18 °C around the dorsoventral compartment boundary, indicating that N signaling was augmented there (Fig. 7D compared with 7C) (Oda & Tsukita 1999). This increase was not due to ectopic proliferation of the wing pouch cells (Supplementary Fig. S3D,E). In these wing discs, the localization of N and Dl extended basally, which may have been responsible for the augmented N signaling (Supplementary Fig. S3B). However, an even higher level of DEcad expression, obtained at 25 °C, partially inhibited N signaling. This was most likely because the epithelial cell morphology was severely disrupted (Fig. 7E, Supplementary Fig. S3C), resulting in the disruption of normal ligand–receptor interactions, and consequently impairing N signaling. In these wing discs, ectopic apoptosis was not detected (data not shown).
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Discussion |
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Polarized vesicular transportation has been reported for many secreted and membrane proteins in epithelial cells (Rodriguez-Boulan et al. 2005). However, the mechanisms of these processes are not well understood. In this study, we showed that N and its ligands predominantly localized to the SAC/AJs in the polarized epithelium of the wing disc. Our results suggest that the localization of N to the SAC/AJs involves two distinct vesicular transportation events (Fig. 8). First, nascent N is transported toward the apical plasma membrane or AJs by polarized exocytosis, which is independent of N O-fucosylation. Second, N is relocated by Dynamin- and Rab5-dependent transcytosis to the SAC/AJs, and this step depends on a novel function of N O-fucosylation by O-fut1.
In the first step of this N transportation, N is delivered by polarized exocytosis. Although the mechanisms of polarized exocytosis remain largely unknown, the polarized transportation of vesicles to the apical membrane proteins is reported to involve a tetanus-insensitive -SNARE and the syntaxin family (Galli et al. 1998; Low et al. 1998). Unexpectedly, N was not delivered to the SAC by polarized exocytosis, although most of the N was localized there, but N was efficiently transported to the other apical regions, in these cells. Therefore, our results suggest that polarized exocytosis could be regulated very precisely when post-Golgi transport vesicles select their targets. The mechanisms responsible for the observed preferential transportations are presently unknown. One possible explanation for this preferential vesicle transportation is that docking and fusion machineries for the N-containing vesicles may be localized to highly specific regions with respect to apicobasal polarity. However, the polarized exocytosis of N is not sufficient for its localization to the SAC/AJs, because N's exocytosis was not disrupted in O-fut1– cells, even though O-fut1 is required for N localization to the SAC/AJs. In our analyses, it was difficult to detect the fusion of N-containing vesicles with the plasma membrane. However, our live cell labeling with an anti-N antibody supported our idea that this polarized exocytosis occurred normally in the O-fut1– cells. It was previously reported that knocking down O-fut1 by RNA interference results in the accumulation of N in the ER (Okajima et al. 2005). However, our higher-resolution studies, which used a deconvolution analysis, revealed that N did not accumulate in the ER in O-fut1– cells, although we failed to determine the nature of the subcellular compartment in which it did accumulate (T.S., I.H.O., N.S., Syunsuke Higashi, M.K., Shiho Nakao, Tomonori Ayukawa, Toshiro Aigaki, Katsuhisa Noda, Eiji Miyoshi, Naoyuki Taniguchi, & K.M., submitted). Therefore, future studies are required to determine where N accumulates in O-fut1– cells.
The second step of N transportation, the re-localization of N from the apical region of the plasma membrane to SAC/AJs, was found to require transcytosis. In one of the rare other examples of this, it was reported that GPI-anchored proteins are delivered from the basolateral surface to the apical surface by transcytosis (Polishchuk et al. 2004). Our results suggest that the absence of O-fucosylation on N does not influence N's exocytosis to the plasma membrane but disrupts its transcytosis. If this is the case, one might expect N to accumulate at the apical plasma membrane in Gmd– or O-fut1– cells. However, we did not observe such an accumulation of N in these cells: N was incorporated normally by endocytosis in Gmd– cells (T.S., I.H.O., N.S., Syunsuke Higashi, M.K., Shiho Nakao, Tomonori Ayukawa, Toshiro Aigaki, Katsuhisa Noda, Eiji Miyoshi, Naoyuki Taniguchi, & K.M, submitted). Thus, N is probably removed from the apical membrane and degraded by the endocytic pathway, so that the amount of N at the cell surface is maintained at the normal level in these cells.
Because mutant N proteins lacking the EGF repeats failed to localize to SAC/AJs (our unpublished data), O-fucosylation of some of the EGF repeats in the extracellular domain of N is probably required for this process. O-glycans are reported to function as apical sorting signals (Yeaman et al. 1997; Alfalah et al. 1999). Thus, it is possible that a similar mechanism occurs in the O-fucosylation-dependent transcytosis of N.
Apicobasally polarized localization of juxtasignaling receptors and ligands may be involved in their signal transduction
We demonstrated that DEcad was required for the localization of N and its ligands to the SAC/AJs. Because the loss of DEcad results in the disruption of apical polarity (Bilder et al. 2003; Tanentzapf & Tepass 2003), the delivery of N to SAC/AJs probably depends on the apical polarity of the epithelial cells. We found that the knock down of DEcad suppressed both N and Fat signaling. Fat, a transmembrane receptor, and its transmembrane ligands are also localized to the apical portion of the wing disc epithelium (Ma et al. 2003). Thus, we speculate that the apicobasally polarized localization of juxtasignaling receptors and/or ligands is a general mechanism required for the activation of their signaling. One simple explanation for this is that receptors and their ligands must confront one another between adjacent cells at the same apicobasal level to carry out their extracellular interactions. However, it is also possible that the SAC/AJs provide a specific environment that is essential for the ligand-dependent activation of N.
N signaling plays essential roles in various types of cells, including epithelial, mesenchymal (Saga & Takeda 2001), and neuronal cells (Artavanis-Tsakonas et al. 1999). The N localization to the SAC/AJs is probably a specific requirement for N signaling in epithelial cells. We speculate that the first step is generally required for cell types. The second transportation step may be different for different cell types, assuring that ligand–receptor interactions occur at a cell-type-specific subcellular location.
In vertebrates, the hallmark structures of apicobasal polarity are different from those of invertebrates (Müller 2000; Knust & Bossinger 2002). However, the structure and components of the SAC and AJs are conserved between vertebrates and invertebrates (Knust & Bossinger 2002). Therefore, it is possible that the apico-basally polarized localization of N is also required for vertebrate N signaling, which may introduce a novel prospect for studying its regulation.
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Experimental procedures |
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Flies were cultured on standard food at 25 °C or 18 °C, as indicated in the figure legends. We used Canton-S as the wild-type strain, O-fut14R6 as the O-fut1 mutant, Notch54l9 or Notch55e11 as the Notch mutant, and DlRev10 SerRX106 as the double mutant of Dl and Ser. GmdH78, a mutant of Gmd, was generated by imprecise excision of a P-element from GmdGS13045 (Toba et al. 1999). GmdH78 lacks most of the Gmd-coding exons (
0.8 kb) located 3' of the P-element. DEcad RNA interference (RNAi) lines carry a pUAST-R57 vector containing an insertion of an inverted repeat (IR) of DEcad cDNA (nucleotides 26-525 of the coding sequence (Pili-Floury et al. 2004). We used the following UAS lines: UAS-shotgun (shg) IR for DEcad RNAi, UAS-DEFL for DEcad over-expression (UAS-shg) (Oda & Tsukita 1999), and UAS-DIAP1 for blocking apoptosis (Kuranaga et al. 2002). These UAS constructs were driven by MS1096-Gal4 and/or patched (ptc)-Gal4 drivers in the wing discs of third-instar larvae (Flybase: http://flybase.bio.indiana.edu/). hsp70-N+-GV3 was described previously (Struhl & Adachi 1998).
Generation of mutant mosaics and time-course analysis involving nascent N
Somatic mosaics were generated using the FLP/FRT system by a 60-min HS at 37 °C in second-instar larvae (Xu & Rubin 1993). To make a mutant clone of O-fut14R6 in wing discs expressing nascent N, w; FRTG13 Ubi-GFP; N+-GV3/TM6B virgin females were crossed with y w hs-flp/Y; FRTG13 O-fut14R6/CyO. The N+-GV3 was expressed by a 15-min HS at 37 °C in late third-instar larvae.
Detection of cell-surface N in living epithelial cells
To detect cell-surface N, dissected wing discs were incubated in rat1, an antibody against the extracellular domain of N, diluted 1/100 in M3 medium (SIGMA), at 4 °C for 2 h. The discs were rinsed 4 times with M3 and once with PBS at 4 °C, then fixed and stained as described above.
Blocking of endocytosis by shiTS1 and dominant-negative Rab5
shiTS1 were cultured at 18 °C, and third-instar larvae were collected and raised at the non-permissive temperature (32 °C) in a water bath for the times indicated in the figure legends. To inhibit the activity of Rab5 in a temperature-sensitive manner, a dominant-negative form of Rab5 (Rab5DN) was expressed from UAS-Rab5 N142I under the control of ptc-Gal4 and a temperature-sensitive Gal80 (Gal80ts), a suppressor of Gal4, using the TARGET method (Shimizu et al. 2003; McGuire et al. 2004). This allowed Rab5DN expression at 32 °C but not 18 °C.
Image scanning and processing
Immunostained wing discs were analyzed by acquiring serial optical sections on a Carl Zeiss LSM 510 META and LSM 5 PASCAL confocal microscope. The images shown in Figs 1–6, S1, S2, and S3 were deconvoluted using AutoDeblur software (AutoQuant). The images shown in Fig. 7 were combined by maximum projection using LSM 5 Image Examiner software from Zeiss.
Immunohistochemistry
Wing imaginal discs dissected from third-instar larvae were stained as described previously (Matsuno et al. 2002). The following antibodies were used: mouse anti-N [C17.9C6 Developmental Studies Hybridoma Bank (DSHB)], 1:100; rat anti-N (rat1 provided by S. Artavanis-Tsakonas), 1:1000; mouse anti-Dl (C594.9B DSHB), 1:100; guinea pig anti-Dl (GP581 provided by M. A. Muskavitch), 1:3000; rat anti-Ser (provided by K.D. Irvine), 1:1000; rabbit anti-nPKC
polyclonal (C20 Santa Cruz Biotechnology), 1:200; rat anti-DECad (DCAD2 DSHB), 1:200; mouse anti-Arm (N2 7A1 DSHB), 1:10; mouse anti-Dlg (4F3 DSHB), 1:10; guinea pig anti-Cora (provided by R.G. Fehon), 1:2500; mouse anti-GAL4 (DBD) (RK5C1 Santa Cruz Biotechnology), 1:100; mouse anti-Wg (4D4 DSHB), 1:100; mouse anti-Cut (2B10, DSHB), 1:100; rabbit anti-Cleaved Caspase-3 (Asp175) (Cell Signaling), 1:200; rabbit anti-GFP (MBL), 1:1000; rabbit anti-phospho-Histone H3 (Upstate), 1:500; rabbit anti-Cis Golgi (GM130 provided by S. Goto), 1:400; Guinea Pig anti-Hrs (provided by H.J. Bellen), 1:400; and rat anti-Rab11 (provided by R.S. Cohen) 1:400. For detection, FITC- (Jackson Laboratories), Alexa 488- (Molecular Probes), Cy3- (Jackson Laboratories), and Cy5- (Rockland) conjugated secondary antibodies were used at 1:200. F-actin labeling was performed after immunostaining using rhodamine phalloidin (Molecular Probes) at a 1:10 dilution for 1 h at room temperature.
Immunoprecipitations and immunoblotting
The immunoprecipitation procedure was described previously (Kitagawa et al. 2001). The primary antibodies used for blotting were anti- nPKC polyclonal (C10), anti-DEcad (DCAD2), anti-Dlg (4F3), anti-Notch (9C6), anti-Delta (9B), and anti-beta tubulin (E7). In some cases, the membranes were stripped in 2% SDS, 100 nM 2-Mercaptoethanol, and 62.5 nM Tris–HCl (pH 6.8) buffer, and reprobed.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: matsuno{at}rs.noda.tus.ac.jp
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References |
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