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1 Department of Biology, Faculty of Science, and Graduate School of Science and Technology, Chiba University, Yayoicho, Inageku, Chiba, Chiba 263-8522, Japan
2 CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
3 Department of Microbiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
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Abstract |
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Introduction |
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M-Ras was first identified as the protein that participates in actin filament reorganization to induce microspikes in cultured cells (Matsumoto et al. 1997). It also brings about in fibroblasts dendritic appearances reminiscent of differentiated neuronal cells (Matsumoto et al. 1997). Different from most of the other Ras family proteins, which are ubiquitously expressed in a variety of tissues and cells, M-Ras is predominantly expressed in brain and less abundantly in skeletal muscle, heart (cardiac muscle), and cultured skeletal muscle myoblasts (Matsumoto et al. 1997; Ehrhardt et al. 1999; Louahed et al. 1999). These findings suggest that M-Ras primarily exerts neuronal functions. M-Ras also shares several effector proteins with Ras. It does not mediate activation of ERK pathway in mouse fibroblasts but preferentially activates PI3K signaling (Kimmelman et al. 2000). By contrast, M-Ras activates ERK pathway by activating B-Raf in PC12 cells stimulated with nerve growth factor (NGF) (Kimmelman et al. 2002).
The rat pheochromocytoma-derived PC12 cell line (Greene & Tischler 1976) provides a model system for growth and differentiation of neuronal cells. PC12 cell differentiation was brought about by the treatment with NGF (Greene & Tischler 1976), 3',5'-cyclic AMP (cAMP) analogs (Schubert et al. 1977), or pituitary adenylate cyclase-activating polypeptide (PACAP) (Deutsch & Sun 1992). All these reagents are likely to induce differentiation through activating the ERK pathway (Vaudry et al. 2002). Among them, NGF activates ERK pathway transiently and continuously through activating Ras family proteins (Qui & Green 1992; Marshall 1995). The transient activation of ERK pathway is not sufficient for the neuronal differentiation and its sustained activation is required (Qui & Green 1992; Marshall 1998). NGF transiently activates Ras, and consequently Ras induces transient activation of ERK pathway. In contrast, it has been reported that NGF causes sustained activation of Rap1 and that activated Rap1 induces sustained activation of B-Raf and subsequent ERK pathway (York et al. 1998). Thus, the sustained activation of ERK pathway mediated by Rap1 has been postulated to be required for the neuronal differentiation of PC12 cells (York et al. 1998).
Constitutively activated mutants of Ras, which can cause sustained activation of ERK pathway, efficiently bring about neuronal differentiation. However, reported results of the effects of a constitutively activated Rap1 mutant on PC12 cell differentiation are contradictory. One report has shown that the activated Rap1 mutant induces neurite outgrowth (Vossler et al. 1997), whereas another report has shown that the identical Rap1 mutant is incapable of inducing neuritogenesis (Bouschet et al. 2003).
On the other hand, exogenous expression of a constitutively active M-Ras induces neurite outgrowth in PC12 cells (Kimmelman et al. 2002). Furthermore, NGF stimulation activates M-Ras, and activated M-Ras binds B-Raf and induces ERK pathway (Kimmelman et al. 2002). However, these results do not discriminate M-Ras from Ras or Rap1 in the property of ERK pathway activation and do not clarify the question whether M-Ras is actually required apart from Ras and Rap1 for PC12 cell differentiation. Thus, we addressed whether M-Ras exerts specific functions in PC12 cell differentiation and whether it is essential for the differentiation. NGF signaling induced rapid and sustained activation of M-Ras leading to sustained activation of ERK pathway and neuritogenesis. In contrast, NGF signaling only transiently activated all three Ras proteins. Exogenously expressed Rap1 caused neither activating phosphorylation of ERK nor neuritogenesis. Accordingly, we conclude that M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway.
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Results |
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Oncogenic or constitutively active mutants of Ras proteins have been shown to induce neuronal differentiation represented by neurite outgrowth in PC12 cells (Bar-Sagi & Feramisco 1985; Noda et al. 1985). To assess the possibility of involvement of M-Ras in PC12 cell differentiation, Myc-epitope-tagged constitutively active mutant M-Ras(G22V) (Gly22 is converted to Val) was expressed in PC12 cells by transfection. Similarly, Myc-tagged constitutively active H-Ras(G12V) and Rap1(G12V) were also expressed in the cells. About 60% of the cells expressing M-Ras(G22V) extended long neurites (neurites more than two-fold longer than the cell body) by 48 h after the transfection (Fig. 1A,B). The efficiency of neuritogenesis and the length of neurites in these cells were almost comparable to those in H-Ras(G12V)-expressing cells (Fig. 1A,B). Notably, Rap1 (G12V)-expressing cells barely extended neurites as did control cells (Fig. 1A,B), consistent with the previous report (Bouschet et al. 2003).
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Endogenous M-Ras is required for NGF-induced neuritogenesis
Next, to examine whether M-Ras was involved in NGF-induced neuronal differentiation, PC12 cells were transfected with Myc-tagged dominant–negative M-Ras(S27N) and stimulated with NGF for 72 h. Although approximately 50% of control cells extended long neurites after treatment with NGF, only approximately 17% of the cells expressing M-Ras(S27N) generated long neurites after NGF stimulation (Fig. 2A,B). The degree of suppression of neuritogenesis by M-Ras(S27N) was similar to that by dominant–negative H-Ras(S17N) (Fig. 2A,B). Moreover, dominant–negative Rap1(S17N) also interfered with the NGF-induced neuritogenesis (Fig. 2A,B). These results suggest that not only H-Ras but also M-Ras is required for NGF-induced neuritogenesis in PC12 cells. In contrast, the prevention of neuritogenesis by the dominant–negative Rap1 might be caused indirectly by suppressing integrin-mediated cell adhesion or by some other mechanism (see Discussion).
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NGF signaling-activated M-Ras and H-Ras induce neuritogenesis by activating ERK pathway
From the above results, NGF signaling is expected to activate M-Ras as well as H-Ras. In addition, since cAMP signaling also induces PC12 cell differentiation possibly through a Ras family protein (Vaudry et al. 2002; Bos 2003), we examined whether NGF and cAMP stimulation activated M-Ras and H-Ras in PC12 cells. To detect the activation of M-Ras and H-Ras, we analyzed their binding to Nore1, which is an effector protein of both Ras and M-Ras (Ortiz-Vega et al. 2002). Both M-Ras(G22V) and H-Ras(G12V) but not their wild-type proteins strongly bound to Nore1 in pull-down assay (Fig. 3A). When PC12 cells were stimulated with NGF, both M-Ras and H-Ras were activated as shown by the binding to Nore1 (Fig. 3B). Stimulation with dibutyryl cAMP (dbcAMP), a plasma membrane-permeable cAMP analog, however, did not activate them. Thus, both M-Ras and H-Ras are activated by NGF signaling but are unlikely to be activated by cAMP signaling.
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M-Ras and H-Ras induce neuritogenesis by activating CREB
The signaling of NGF–ERK pathway leads to cAMP response element (CRE)-mediated transcription through the activating phosphorylation of the transcription factor CREB and its coactivator CBP (Shaywitz & Greenberg 1999; Lonze & Ginty 2002; Vaudry et al. 2002). Thus, we examined whether M-Ras-mediated neuritogenesis required phosphorylation of CREB and its transcriptional activity. Although most of the control PC12 cells had unphosphorylated inactive CREB, the cells transfected with M-Ras(G22V) as well as those with H-Ras(G12V) had CREB with the activating phosphorylation in approximately 80% of the nuclei (Fig. 4A,B). Then, to assess whether this phosphorylation of CREB led to the activation in its transcriptional activity, we analyzed the activity of CRE-mediated transcription by luciferase assay. Both M-Ras(G22V) and H-Ras(G12V) activated the CRE-mediated luciferase activity approximately 4.5-fold (Fig. 4C). The addition of U0126 or another MEK inhibitor PD98059 reduced both the M-Ras(G22V)- and H-Ras(G12V)-induced activities to approximately 50%, whereas the treatment with SB202190 or PI3K inhibitor wortmannin did not affect the activities (Fig. 4C). Therefore, both continuously activated M-Ras and H-Ras can induce CREB activation by phosphorylating it. The activation is mediated by ERK pathway but not by p38 pathway or PI3K signaling.
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Both M-Ras and H-Ras are expressed in the central nervous system
The results described above do not distinguish M-Ras from H-Ras in the properties that they are activated by NGF signaling but not by cAMP signaling, that they activate ERK pathway and transcriptional activity of CREB, and that they induce neuritogenesis if they are continuously activated. Thus, we addressed whether they were discriminated by the difference in distributing areas in nervous system. In situ hybridization in adult mouse brain showed that M-Ras was highly expressed in the entire region of the hippocampus and in the cerebellum (Fig. 5Aa). In the cerebellum, M-Ras was expressed most highly in Purkinje cells and at a moderate level in the granular layer (Fig. 5Ab). M-Ras was also abundantly present in the gray matter of the spinal cord in both anterior and posterior regions (Fig. 5Ba,c). H-Ras was similarly highly expressed in the hippocampus, cerebellum and the gray matter of the spinal cord (Fig. 5Ad,e,Be,g). Accordingly, M-Ras and H-Ras are similar in their distribution in the central nervous system.
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Although NGF stimulation has been reported to bring about sustained activation of Rap1 and ERK pathway (York et al. 1998), the expression of the constitutively active Rap1(G12V) caused neither ERK phosphorylation nor neurite extension as shown above. Thus, we assessed whether NGF induced sustained activation of M-Ras leading to neuritogenesis in PC12 cells. In PC12 cells with serum starvation for 16 h, the activities of M-Ras and all the Ras proteins were at very low or undetectable levels as analyzed by the pull-down assay with GST–Nore1 and antibodies specific for M-Ras, H-Ras, K-Ras, and N-Ras, respectively (Fig. 6A). NGF treatment of the serum-starved cells resulted in rapid activation of M-Ras as well as H-Ras, K-Ras and N-Ras within 2 min after the stimulation (Fig. 6A). The activities of H-Ras, K-Ras, and N-Ras precipitously declined by 5 min and became undetectable or very low levels between 10 min and 6 h. Their activities were slightly elevated 12 h after the stimulation and maintained for > 12 h after the elevation. By contrast, the activity of M-Ras was steadily sustained for > 24 h after the initial activation (Fig. 6A). The activating phosphorylation of ERK1/2 and that of CREB in the serum-starved cells were barely detectable as detected by immunoblotting with pAbs to phospho-ERK and phospho-CREB, respectively (Fig. 6A). NGF treatment of the cells caused the phosphorylation of ERK1/2 and CREB within 2 min after the stimulation (Fig. 6A). Their phosphorylation reached maximum levels around 5 min after the stimulation and was maintained at relatively high levels for > 1 h. After that, the phosphorylation levels declined to some degree but were sustained at the lower levels for > 24 h.
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Discussion |
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Although the constitutively activated Rap1 did not induce neuritogenesis, the dominant-negative Rap1 as well as the dominant-negative M-Ras and H-Ras suppressed NGF-induced neuritogenesis. This is reminiscent of the finding that the expression of a constitutively active Cdc42 does not cause neurite extension in PC12 cells but that a dominant-negative Cdc42 interferes with NGF-induced neuritogenesis (Abe et al. 2003). Cdc42 is essential for filopodial or lamellipodial formation at the growth cone of neurites, which is required for neurite extension (Kozma et al. 1997; Abe et al. 2003). Nevertheless, because the formation of these structures on the growth cone is not sufficient for the induction of neuritogenesis, the expression of Cdc42 may not lead to neuritogenesis. On the other hand, Rap1 plays important roles in both integrin-mediated cell–substrate adhesion and cadherin-mediated cell–cell adhesion (Caron 2003; Bos et al. 2003; Bos 2005). Rap1 regulates all integrins that are associated with the actin cytoskeleton, i.e. ß1, ß2, and ß3 family integrins. ß1 and ß3 integrins and integrin-linked kinase (ILK) that interacts with these integrins are required for the NGF-induced neuritogenesis of PC12 cells (Zhang et al. 1993; Yip & Siu 2001; Mills et al. 2003). Thus, the dominant-negative Rap1 might interfere with the neuritogenesis by suppressing the expression or functions of these integrins (Fig. 7).
Recently, Yamada et al. (2005) have shown that Rap1 induces neuritogenesis in mouse neuroblastoma NG108 cells by activating a downstream target protein RA-RhoGAP, which inactivates RhoA. RhoA and its target protein ROCK cause neurite retraction, and consequently inactivation of RhoA leads to neurite extension. A cAMP analog 8CPT-cAMP induces the Rap1-mediated neuritogenesis in this cell line (Yamada et al. 2005). However, this cell line does not respond to NGF for neuritogenesis (Mutoh et al. 2002). In the case of PC12 cells, RhoA and ROCK also induce neurite retraction (Katoh et al. 1998), and NGF suppresses RhoA activity (Yamaguchi et al. 2001). This NGF-induced inactivation of RhoA is rapid and transient, however, and it is uncertain whether the NGF-induced RhoA inactivation is responsible for neuritogenesis. Thus, it remains to be examined whether the Rap1-mediated inactivation of RhoA is applicable for NGF-induced neuritogenesis in PC12 cells.
The distribution of M-Ras and H-Ras expression in central nervous system detected by in situ hybridization was basically indistinguishable. However, M-Ras has a polybasic domain and lacks palmitoylation sites in C-terminal hypervariable region as does K-Ras4B (Matsumoto et al. 1997), whereas H-Ras has two palmitoylation sites and lacks a polybasic domain. These membrane-interacting modules are likely to determine the membrane trafficking pathway from the endoplasmic reticulum to the plasma membrane and the localization on microdomains of the plasma membrane as well as on the endomembranes (Hancock 2003). The differences in localization of M-Ras and Ras proteins may regulate their accessibility to different types of receptors, GEFs, GTPase-activating proteins (GAPs), and effector proteins. For example, the Ras GEF Sos is phosphorylated by ERK, resulting in uncoupling Sos from Grb2, which is an adaptor protein between NGF receptor TrkA and Sos. This mechanism has been proposed to provide a negative feedback to limit Ras activation (Corbalan-Garcia et al. 1996). Alternatively, recruitment of p120 RasGAP to TrkA via the docking protein Dok may be responsible for the limited activation of Ras (Sasagawa et al. 2005). M-Ras has been shown to be activated by the GEF RasGRP, which may not be subjected to negative feedback from ERK, through NGF stimulation in PC12 cells (Kimmelman et al. 2002). In addition, since IQGAP1 has been tentatively identified as a GAP for M-Ras (Vasilescu et al. 2004), p120 RasGAP might not exert GAP function on M-Ras. In this consequence, NGF stimulation might result in the transient activation of Ras and sustained activation of M-Ras, both of which are presumably required for neuronal differentiation of PC12 cells. Therefore, Ras and M-Ras seem to function cooperatively but in a temporally and spatially distinct manner for NGF-induced neuronal differentiation and possibly for some neural functions in central nervous system as well.
PACAP-induced cAMP signaling activates both protein kinase A (PKA) and ERK pathway and leads to neuritogenesis in PC12 cells (Barrie et al. 1997; Lazarovici et al. 1998; Vaudry et al. 2002). cAMP-mediated activation of PKA has been shown to activate B-Raf and subsequent ERK pathway through activating Rap1 by phosphorylation (Vossler et al. 1997). On the contrary, other reports have shown that cAMP-induced regulation of ERK pathway and activation of Rap1 are independent processes (Busca et al. 2000; Enserink et al. 2002; Christensen et al. 2003). They argue that cAMP-induced activation of Rap1 is mediated by Epac, the cAMP-activated GEF for Rap1, and that cAMP-induced activation of ERK pathway is mediated by PKA in a manner that is independent of Rap1 (Enserink et al. 2002; Bos 2003; Christensen et al. 2003). So far, the mechanism by which cAMP activates B-Raf and ERK pathway has been open to question and controversial (Bos 2003; Stork 2003). Thus, we examined whether M-Ras and H-Ras were involved in the cAMP-induced ERK pathway and neuritogenesis, but neither M-Ras nor H-Ras was activated by dbcAMP stimulation. Consequently, both M-Ras and H-Ras are likely to play their roles primarily in NGF-induced but not in PACAP-induced PC12 cell differentiation or in crosstalk between cAMP signaling and ERK pathway in the differentiation. Other members of Ras family or protein kinase C might participate in the cAMP-mediated ERK pathway (Barrie et al. 1997; Lazarovici et al. 1998; Bouschet et al. 2003).
NGF-induced transient and sustained activation of ERK pathway phosphorylates the transcription factor, ternary complex factor (TCF), in PC12 cells. The phosphorylated TCF coupled with another transcription factor, serum-response factor (SRF), causes neuritogenesis by expressing p35, which is the neuron-specific activator of Cdk5, through inducing the transcription factor Egr1 (Harada et al. 2001). We showed here that the neuritogenesis brought about by M-Ras as well as by H-Ras requires activation of CREB by phosphorylation and CRE-mediated transcription, which is dependent on ERK pathway. Furthermore, NGF stimulation induced sustained phosphorylation of CREB. The sustained phosphorylation of CREB is likely to be caused by the M-Ras-mediated sustained activation of ERK pathway presumably activating p90 ribosomal S6 kinase (RSK) (Xing et al. 1998; Silverman et al. 2004) (Fig. 7). The TCF-mediated transcriptional activity and the CREB-mediated transcriptional activity may induce transcription of subsets of genes distinct from each other, but both of them may be indispensable for the neuritogenesis of PC12 cells. Therefore, M-Ras seems to serve as the master regulator of the neuritogenesis by continuously activating both these transcriptional pathways.
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Experimental procedures |
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The rat pheochromocytoma PC12 cells (Greene & Tischler 1976) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5% horse serum. To induce differentiation, the medium was replaced with DMEM supplemented with 100 ng/mL NGF (2.5 S, Promega) or with 10% FBS, 5% horse serum, and 0.5 mM dbcAMP (Sigma). The degree of neuritogenesis was analyzed by measuring the length of neurites as a multiple of cell body diameter. Cells with neurites > two-fold longer than the cell body diameter were regarded as fully differentiated cells.
Epitope-tagging, transfection and fluorescence microscopy
cDNAs of mouse H-Ras and Rap1A were cloned from the mouse C2 myoblast and myotube cDNA libraries constructed in 
ZAPII (Matsumoto et al. 1997). Point mutations to generate the constitutively active mutants H-Ras(G12V) and Rap1(G12V) and the dominant-negative mutants H-Ras(S17N) and Rap1(S17N) were introduced in their cDNAs with Transformer site-directed mutagenesis kit (BD Biosciences Clontech). These mutant cDNAs and the cDNAs of constitutively active rat M-Ras(G22V) and dominant-negative M-Ras(S27N) (Matsumoto et al. 1997) were inserted into the pEF-BOS/Myc vector (Mizushima & Nagata 1990) in frame with the Myc-tag sequence. Human CREB cDNA was subcloned in the CMV promoter-driven pCG vector. A dominant–negative KCREB (Walton et al. 1992) cDNA was ligated into the pCG-N vector in frame with the influenza virus hemagglutinin (HA)-tag sequence. These recombinant plasmids were transfected to PC12 cells grown on glass coverslips by the calcium phosphate-mediated method as previously described (Endo et al. 1996). The transiently transfected cells were processed for immunofluorescence microscopy 48 h after the transfection (Endo & Nadal-Ginard 1998). The fixed and permeabilized cells transfected with Myc- and HA-tagging constructs were incubated with the monoclonal antibody (mAb) Myc1-9E10 (Evan et al. 1985) (American Type Culture Collection) and anti-HA rabbit pAb (MBL), respectively. To detect activating phosphorylation of ERK1/2 and CREB, the cells were incubated with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) pAb and anti-phospho-CREB (Ser133) pAb (Cell Signaling Technology), respectively. They were then incubated with fluorescein isothiocyanate- or rhodamine-conjugated goat anti-mouse or anti-rabbit IgG (affinity-purified, Cappel). The specimens were observed with a Zeiss Axioskop microscope equipped with phase-contrast and fluorescence optics.
Preparation of anti-M-Ras antibody
Rat Mras cDNA was ligated into pQE-30 vector (Qiagen) in frame with 6 x His-tag sequence. The His-tagged recombinant M-Ras was expressed in the E. coli strain XL1-Blue and affinity-purified with Talon Metal Affinity Resin (BD Biosciences Clontech). A New Zealand white rabbit was immunized with the purified M-Ras protein emulsified with Freund's complete and incomplete adjuvant (Difco Laboratories). The anti-M-Ras pAb was affinity-purified through HiTrap HNS-activated (Amersham Biosciences) coupled with the antigen.
RNAi
RNAi of M-Ras was conducted with pSilencer 2.1-U6 neo siRNA expression vector (Ambion). The target sequences of rat Mras were 5'-GCATACAGAGATCGACAAT-3' (sequence 1: nucleotides 156–174 from the initiation codon) and 5'-CAAAGTGGACCTGATGCAC-3' (sequence 2: nucleotides 378–396 from the initiation codon). A pSilencer plasmid encoding a validated non-targeting siRNA (Ambion) was used as a negative control. The rat skeletal muscle L6E9-B cells (Endo & Nadal-Ginard 1987) were stably transfected with the pSilencer plasmids by using FuGENE 6 Transfection Reagent (Roche Applied Science), and stable transfectants were selected with G418. The amounts of M-Ras and classical Ras proteins were detected by immunoblotting with the anti-M-Ras pAb and anti-pan-Ras mAb (Oncogene Research Products), respectively. PC12 cells were co-transfected with the pSilencer plasmids and one-tenth of the amount of pEGFP-C1 vector (BD Biosciences Clontech) to monitor the siRNA-expressing cells. Twenty-four hours after the transfection, the cells were treated with NGF for 72 h.
Pull-down assay
Pull-down assay was conducted as previously described (Abe et al. 2003). The cDNA encoding Nore1 was cloned from BD Matchmaker mouse brain cDNA library (BD Biosciences Clontech) by yeast two-hybrid screening using M-Ras(G22V) cDNA in pGBT9 vector as a bait. The Nore1 cDNA fragment encoding amino acids 171–413 was ligated into the pGEX-2T vector (Amersham Biosciences) in frame with glutathione S-transferase (GST)-tag sequence. The GST-tagged recombinant protein was expressed in E. coli and affinity-purified with glutathione–Sepharose 4B (Amersham Biosciences). PC12 cells transfected with pEF-BOS/Myc-M-Ras or pEF-BOS/Myc-H-Ras or the cells stimulated with NGF were lysed with the lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1% Nonidet P-40, 1 mM 2-mercaptoethanol, 0.1 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 1 µg/mL pepstatin). The lysates were incubated with the recombinant GST–Nore1 coupled to glutathione–Sepharose 4B for 2 h at 4 °C and extensively washed with the lysis buffer. The binding proteins were eluted with the dissociation buffer (20 mM Tris–HCl, pH 7.5, 20 mM EDTA, and 2% SDS) and analyzed by immunoblotting with either the anti-Myc mAb or the anti-M-Ras pAb, anti-H-Ras, anti-K-Ras, and anti-N-Ras mAbs (Oncogene Research Products) after SDS-polyacrylamide gel electrophoresis.
Luciferase assay
The plasmid pGL3/TK-CRE contains five tandem repeats of CRE sequence ligated with thymidine kinase promoter–luciferase gene. PC12 cells were transfected with pGL3/TK-CRE and pMiwZII, a lacZ expression vector, together with pEF-BOS/Myc-M-Ras(G22V) or pEF-BOS/Myc-H-Ras(G12V) by using SuperFect Transfection Reagent (Qiagen). The cells were treated with 20 µM U0126 (Promega), 50 µM PD98059 (Promega), 1 µM SB202190 (Calbiochem), or 1 µM wortmannin (Sigma) 3 h after the transfection. They were lysed with the Reporter Lysis Buffer (Promega) 24 h after the transfection. Ten microliters of the cell lysates were added to 100 µL of Luciferase Assay Reagent (Promega), and luciferase activity was analyzed with a luminometer Flash’n Glow LB955 (Perkin Elmer Life Science). The CRE sequence-mediated luciferase activity was normalized for the transfection efficiency with the ß-galactosidase activity, which was assessed by using ß-Galactosidase Enzyme Assay System (Promega).
In situ hybridization
Six-week-old mice were fixed by perfusion of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Cryosections (14 µm thick) of the brain and spinal cord were prepared with Leica Cryomicrotome CM1850. Fragments of the mouse Mras cDNA (nucleotides +1 to +563, where the first nucleotide of the initiation codon is +1) and the mouse Hras cDNA (–68 to +570) were inserted into pBluescript II KS(–) (Stratagene). In vitro transcripts were synthesized with T3 and T7 polymerases (Promega) in the presence of digoxigenin-11-UTP (Roche Applied Science). The sense and anti-sense RNA probes were hybridized with the cryosections in the hybridization buffer [50% formamide, 5 x SSC, 2% Blocking Reagent (Roche), 0.1% CHAPS, 5 mM EDTA, 0.1% Triton X-100, 50 µg/mL heparin, 10 µg/mL tRNA, 10 µg/mL salmon sperm DNA, and 1 x Denhardt's solution] for 16–18 h at 60 °C. The hybridized probes were detected by reacting with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) for 1 h followed by a chromogenic reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: t.endo{at}faculty.chiba-u.jp
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Received: 24 March 2006
Accepted: 13 June 2006
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