SCUBE2 [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein 2] belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by nine EGF-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C-terminus. Despite several genetic analyses suggesting that the zebrafish orthologue of the mammalian SCUBE2 gene participates in HH (Hedgehog) signalling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signalling remains uninvestigated. In the present study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signalling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with SHH (Sonic Hedgehog) and SHH receptor PTCH1 (Patched-1), and enhance the SHH signalling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel positive component of the HH signal, acting upstream of ligand binding at the plasma membrane. Thus human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumour progression.
- cell-surface protein
- CUB domain
- epidermal growth factor-like repeat (EGF-like repeat)
- signal transduction
- Sonic Hedgehog
The SCUBE [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein] family is composed of three independent gene members, SCUBE1–SCUBE3, and is evolutionarily conserved from zebrafish to humans [1–7]. These proteins share an organized protein domain structure of at least five recognizable motifs: an N-terminal signal peptide sequence, followed by nine copies of EGF (epidermal growth factor)-like repeats, a spacer region, three cysteine-rich motifs and one CUB domain at the C terminus [6–8]. Apart from their common expression in the endothelium, individual SCUBE members appear to have a distinctive expression profile. For example, SCUBE3 is expressed in a restricted fashion, such as in osteoblasts and vascular cells . In contrast, SCUBE2 is expressed in a broader spectrum of tissues or cell types, including embryonic neuroectoderm, heart, lung, testis, renal mesangial cells and fibroblast cells [5,7].
However, the complete full-length cDNA for SCUBE2 has not been isolated and characterized, probably because of the presence of multiple splicing variants, which delete exons coding for the EGF-like repeats or the spacer region [1,7]. On the basis of genetic studies, it was suggested that you, the zebrafish orthologue of the mammalian SCUBE2 gene, acts as a permissive factor for HH (Hedgehog) signalling upstream of SMO (Smoothened), the HH receptor signal component [1–3]. Yet the molecular and biochemical mechanism underlying SCUBE2 action during HH signal transduction remains largely unknown.
The HH signal pathway, originally identified in Drosophila and conserved in vertebrates, is critical for embryonic development of multiple tissues and organs [9,10]. Of three highly conserved mammalian HH family members, SHH (Sonic Hedgehog), IHH (Indian Hedgehog) and DHH (Desert Hedgehog), SHH is the most widely expressed and studied . Misregulation of the SHH signalling pathway has been implicated in many human congenital defects and various cancers . In the absence of HH ligands, its receptor PTCH1 (Patched-1) keeps the pathway inactivated by inhibiting the activation of a second transmembrane signalling component, SMO. Upon the binding of HH ligands to PTCH1, the inhibition of SMO is released to transmit the HH signal [9,10].
The SHH precursor is processed to an active 19-kDa protein, which further undergoes dual lipid modification by palmitate at the N-terminus and cholesterol at the C-terminus . The processing, release, trafficking and turnover of the SHH ligand all need to be tightly regulated in the extracellular environment and/or at the cell surface to ensure proper signalling [12,13]. Recent studies have identified a small number of proteins that bind to SHH and modulate SHH-mediated responses in vertebrates. These include heparan sulfate proteoglycans  and the Shifted protein [15,16] in the extracellular matrix, or megalin (a low-density lipoprotein receptor) , Gas1 (growth-arrest-specific 1) [18–20], the membrane glycoprotein Hip1 (HH-interacting protein 1)  and the type I transmembrane immunoglobulin-like/fibronectin type III repeat domain-containing proteins Cdo and Boc [22–24] on the cell surface. However, it is likely that the full spectrum of extracellular or membrane proteins that can modulate SHH signalling and activity is still not completely revealed.
In the present paper, we report on the isolation and functional characterization of the full-length cDNA coding for human SCUBE2. Our results demonstrate for the first time that human SCUBE2 is a cell-surface glycoprotein and forms a complex with SHH and its receptor PTCH1 to promote the SHH-induced signalling. Thus SCUBE2 represents a novel positive component during the reception of the HH signal, and the gene product may have significant implications for HH-mediated biological processes during development and HH-related human pathology, such as tumour progression.
Anti-FLAG M2 and anti-Myc 9E10 monoclonal antibodies were purchased from Sigma and Covance respectively. SHH and caveolin-1 antisera were from Santa Cruz Biotechnology.
Cloning of full-length human SCUBE2
Because the commercially available cDNA for human SCUBE2 represents a splice variant lacking a portion of the EGF-like repeats and spacer region , we swapped in a cDNA fragment to correct the defective region by a standard molecular biology method. The resulting cDNA encodes a polypeptide composed of an organized protein domain structure consistent with that of its zebrafish orthologue and all other SCUBE protein members (see Figure 1) [1–3,6].
Cell culture, construction of expression plasmids and transfection
HEK (human embryonic kidney)-293T and NIH 3T3 cells were maintained in DMEM (Dulbecco's minimal essential medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C in an atmosphere of 5% CO2. The Shh-Light2 reporter cells were cultured in the above-mentioned DMEM supplemented with 0.4 mg/ml G-418 and 0.15 mg/ml zeocin (Invitrogen). The FLAG-epitope-tagged version of the SCUBE2 expression plasmid was prepared similarly as described . HEK-293T or Shh-Light2 cells were transfected by use of Lipofectamine™ 2000 (Invitrogen) or FuGENE™ HD (Roche) respectively.
Treatment with tunicamycin
Transfected cells were cultured in the absence or in the presence of tunicamycin (5 μg/ml) for 24 h. Cell lysates from each culture were analysed by Western blotting with anti-FLAG M2 antibody.
Immunoprecipitation and Western blot analysis
At 2 days after transfection, cells were washed once with PBS and lysed for 15 min on ice in 0.5 ml of lysis buffer (25 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM EDTA, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 10% glycerol, 1% Triton X-100 and 60 mM octylglucoside, which effectively solubilizes caveolar or raft membrane microdomains). Lysates were clarified by centrifugation at 10000 g for 15 min at 4 °C. Cells lysates were incubated with 1 μg of indicated antibody and 20 μl of 50% (v/v) Protein A–agarose (Pierce) for 2 h with gentle rocking. After three washes with lysis buffer, precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS/PAGE (10% gels), and transferred on to PVDF membranes. The membranes were blocked with PBS (pH 7.5) containing 0.1% gelatin and 0.05% Tween 20 and blotted with the indicated antibodies. After two washes, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 h. After washing the membranes, the reactive bands were visualized using ECL® (enhanced chemiluminescence) (GE Healthcare).
Gli-dependent luciferase reporter assay
Assays for HH-pathway activation in Shh-Light2 cells, an NIH 3T3 cell line stably incorporating Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters, were performed as described in . Briefly, the Shh-Light2 cells (2×105 per well) were seeded into 24-well plates and transfected on the following day with an empty vector or the SHH expression plasmid (100 ng) together with various deletion constructs of SCUBE2 (400 ng) as indicated. After 24 h, the growth medium was changed to low-serum medium (0.5% FBS) and cultured for an additional 2 days. Cells were lysed and processed for firefly luciferase reading using the Dual-Luciferase Reporter Assay System (Promega). Data were expressed as relative luciferase activity by dividing firefly luciferase activity with that of Renilla luciferase.
The RNAi (RNA interference)-mediated silencing experiments
We used the vector-based shRNAs (short hairpin RNAs) generated by The RNAi Consortium  to knockdown the endogenous Scube2 (clones TRCN0000109435–TRCN000010943539) in the Shh-Light2 reporter cell line. Owing to the lack of specific antibody against mouse Scube2 protein, the knockdown efficiency of each shRNA clone was verified by quantitative RT (reverse transcription)–PCR, followed by the Gli-dependent luciferase reporter assay to evaluate the effect of RNAi-mediated silencing of Scube2 in the Shh-Light2 reporter cells as above. In addition, a non-targeting shRNA for GFP (green fluorescent protein) (clone TRCN0000072178) also was used as a control and at least two independent sequences of RNAi for Scube2 were functionally effective, diminishing the possibility that the observed effects were due to off-target effects of RNAi.
SHH–AP (alkaline phosphatase) protein binding assay.
The production of AP-tagged SHH protein and the binding experiments were performed essentially as described previously .
Subcellular fractionation of raft membranes
HEK-293T cells expressing SHH alone or together with various forms of SCUBE2 protein underwent initial extraction with 1% (v/v) Triton X-100, followed by sucrose density gradient centrifugation as described in .
Isolation and expression of the full-length cDNA coding for human SCUBE2
To investigate the function of SCUBE2, we first isolated and characterized the full-length cDNA coding for human SCUBE2 as described in the Experimental section. The open reading frame encoding a polypeptide of 1028 amino acids shares a conserved protein domain organization and overall 66% sequence homology with its zebrafish orthologue [1–3] (Figure 1). Since overexpression of recombinant SCUBE1 or SCUBE3 protein resulted in secreted glycoproteins that could form oligomers tethered on the cell surface [6,7], we first examined whether the overexpressed SCUBE2 protein might also behave in a similar way and, if so, which domain is responsible for its secretion and/or cell-surface expression. A comprehensive panel of mutated human SCUBE2 expression constructs containing specific deletion of the EGF-like repeats, spacer region, cysteine-rich repeats and/or CUB domain was generated (Figure 2A). In addition, we produced two deletion mutants mimicking the nonsense mutation in the ty97 or rw87 allele, which presumably encodes truncated null proteins in zebrafish [1–3]. The FLAG epitope tag was added at the N-terminus for easy detection of these mutated SCUBE2 proteins.
The SCUBE2 deletion proteins were expressed by means of transient transfection in HEK-293T cells. At 2 days post-transfection, the conditioned media and cell lysates underwent Western blot analysis with anti-FLAG M2 antibody. As shown in Figure 2(B), all SCUBE2 deletion constructs produced corresponding recombinant proteins in HEK-293T cells (bottom panel). Furthermore, immunoblotting of the conditioned media from transfected cells showed effective secretion of all expressed products, except for SCUBE2-rw87, D2 and D5 mutant proteins, which lack the spacer region and the cysteine-rich repeats (Figure 2B, top panel). These data suggest that the spacer region and/or the cysteine-rich repeats can direct the expressed SCUBE2 protein through the secretory pathway for secretion, at least when overexpressed in HEK-293T cells.
The same set of transfected cells was detached and stained with anti-FLAG M2 antibody to determine their surface expression by flow cytometry (Figure 2C). SCUBE2-rw87 and D2 deletion mutants were defective in membrane association, but SCUBE2-FL (full-length SCUBE2), ty97, D5, D6 and D7 proteins, which all contain the spacer region, the cysteine-rich repeats and/or the CUB domain, were expressed on the cell surface. Together, our results imply that these domains may play important roles in targeting SCUBE2 for secretion and tethering the molecule on the cell surface.
Of eight putative N-linked glycosylation sites, five motifs are well preserved between human and zebrafish SCUBE2 (Figure 1). Indeed, treatment with tunicamycin, an inhibitor of N-glycosylation, resulted in a decrease in molecular mass of the precursor forms of corresponding SCUBE2 proteins (Figure 3), which indicates that most SCUBE2 is glycosylated when expressed in HEK-293T cells. Consistently, tunicamycin treatment did not alter the apparent molecular mass of mutant SCUBE2-rw87 protein, which contains no N-linked glycosylation motif (Figure 3). Similar results were obtained by treatment with peptide N-glycosidase F to remove the N-linked oligosaccharide chain (results not shown). Together, these data demonstrate that SCUBE2 is N-glycosylated at multiple sites mainly within the spacer region and the cysteine-rich repeats.
Human SCUBE2 interacts with SHH and its receptor PTCH1
In the light of recent genetic studies suggesting that zebrafish SCUBE2 functions upstream in the HH signalling pathway [1–3], we next investigated whether SCUBE2 could biochemically associate with SHH ligand and its receptor PTCH1. HEK-293T cells were transfected with the SHH or PTCH1 expression plasmid either alone (both tagged with the Myc epitope for easy detection) or together with a series of FLAG-tagged SCUBE2 deletion expression constructs (Figure 4). At 2 days after transfection, cell lysates underwent immunoprecipitation with the anti-Myc antibody, and the immunoprecipitates were analysed by immunoblotting with anti-FLAG M2 antibody to determine the protein interactions. As shown in Figure 4(A), immunoprecipitation with SHH protein resulted in co-immunoprecipitation of SCUBE2-FL and D5 mutant protein, which suggests that SCUBE2 indeed forms a complex with SHH and that most binding can be ascribed to the C-terminal CUB domain present in the D5 mutant. Similar experiments using anti-FLAG immunoprecipitation followed by anti-Myc Western blot analysis also demonstrated that PTCH1 protein could specifically interact with the SCUBE2-FL, D5 and D7 mutant protein (Figure 4B), which suggests that both the spacer region and the C-terminal CUB domain could interact with the SHH receptor PTCH1. Most importantly, in vitro pull-down assay demonstrates further that SCUBE2 interacts directly with SHH (Figure 4C).
Human SCUBE2 positively regulates the SHH-initiated signalling
Because of the biochemical interactions between SCUBE2 and SHH/PTCH1, we evaluated further whether SCUBE2 can function to regulate the SHH-mediated signal activity. The effect of ectopic SCUBE2 expression on SHH signalling was assessed in the Shh-Light2 cells, an NIH 3T3-derived cell line that responds to SHH by activation of an integrated Gli-dependent luciferase reporter . The Shh-Light2 cells were transfected with the SHH expression plasmid alone or together with expression vectors encoding SCUBE2-FL, ty97, D5 or D6 for 2 days. Expression of SHH increased the Gli-dependent luciferase activity ∼6-fold more than that in cells transfected with empty vector alone (Figure 5A). Interestingly, co-expression of SCUBE2-FL, but not the other mutant ty97, D5 or D6 proteins, further enhanced the SHH-induced transcriptional activation an additional 2–3-fold in a dose-dependent manner (Figure 5A). Similar results were obtained by the use of parental NIH 3T3 cells transiently transfected with the Gli-responsive luciferase reporter construct (results not shown).
Conversely, two independent SCUBE2-specific sequences of RNAi-mediated silencing of SCUBE2, which effectively reduced the SCUBE2 expression (>50% suppression of the Scube2 mRNA expression compared with the RNAi control), significantly diminished the Gli-dependent reporter activity in response to SHH (Figure 5B). Most importantly, treatment with cyclopamine, the SMO antagonist that directly binds and inhibits SMO activity , completely blocked the SCUBE2-mediated enhancement of SHH signalling (Figure 5C).
To evaluate further the relative contribution of other domains in promoting SHH signalling, we obtained two additional deletion constructs, one lacking both the EGF-like-repeat 9 and a portion of the spacer region (SCUBE2-ΔE9-sp) at the N-terminus and the other removing one cysteine-rich repeat (SCUBE2-ΔCR). The SCUBE2-ΔCR mutant was as effective as SCUBE2-FL in enhancing the SHH-dependent reporter activity, but the SCUBE2-ΔE9-sp mutant was incapable of promoting SHH-induced reporter activity (Figure 6). Thus, although the N-terminal EGF-like repeats or spacer region do not participate directly in SHH binding, these domains are essential for SCUBE2 function in enhancing SHH signalling activity.
SCUBE2 enhances SHH binding to the PTCH1-expressing NIH 3T3 cells
To evaluate further whether SCUBE2 could indeed increase the binding of SHH to PTCH1, we first produced a functional N-terminal fragment of SHH fused to AP (SHH–AP, see Figures 7A and 7B) as described in . As shown in Figure 7(C), binding of SHH–AP protein is evident when compared with the control AP protein on the PTCH1-expressing NIH 3T3 cells. Interestingly, SHH–AP binding is significantly increased when the SCUBE2 protein is expressed in NIH 3T3 cells compared with the vector-transfected cells. In agreement with the Gli-luciferase reporter assay (Figure 5), these data suggest that SCUBE2 could enhance the binding of SHH to its receptor PTCH1 in NIH 3T3 cells.
Localization of SCUBE2 within the caveolin-enriched or raft microdomains
Given the above data showing a biochemical interaction between SCUBE2 and SHH or PTCH1 (Figure 4) and that both the cholesterol-tethered active form of HH and overexpressed PTCH1 proteins have been found in the raft microdomains of the plasma membranes [29,30], we investigated further whether SCUBE2 could also be recruited into these cholesterol-rich microdomains. HEK-293T cells expressing SHH, together with various forms of FLAG-tagged SCUBE2 proteins, were homogenized in the presence of 1% (v/v) Triton X-100, and the cell lysates underwent sucrose density gradient centrifugation. The distribution of SHH or SCUBE2 protein was assessed by immunoblotting with the anti-SHH or anti-FLAG M2 antibody respectively. The SCUBE2-FL, ty97 or D5, but not the D6, mutant co-fractionated with SHH and caveolin-1, a caveolar or raft-associated protein, in the low-buoyancy raft compartment (Figure 8, top panel). Similar results were obtained in NIH 3T3 cells transfected with the expression plasmid encoding SCUBE2-FL or D5 protein respectively (Figure 8, bottom panel).
SCUBE2 interacts with caveolin-1 via its C-terminal CUB domain
Since SCUBE2 co-fractionates with caveolin-1 in the lipid raft compartment, we examined further whether these two proteins can interact biochemically. Co-immunoprecipitation experiments were performed in cells transiently transfected with a variety of SCUBE2 deletion constructs together with the caveolin-1 expression plasmid. As shown in Figure 9, immunoprecipitation of caveolin-1 resulted in the co-association of SCUBE2-FL or D5 mutant protein. These results suggest that SCUBE2 forms a complex with caveolin-1, mainly through the C-terminal CUB domain (Figure 9).
Despite several genetic studies implying the involvement of the zebrafish orthologue of the mammalian SCUBE2 gene in the HH signal pathway [1–3], the biochemical or molecular basis for SCUBE2 function remains elusive. In the present study, to uncover the molecular mechanisms by which SCUBE2 modulates the HH-initiated responses, we isolated the full-length cDNA and studied the function of human SCUBE2. Protein sequence analysis showed that human SCUBE2 shares not only high sequence homology, but also an organized domain structure characteristic of the SCUBE protein family, of at least five motifs: an N-terminal signal peptide sequence, nine tandem repeats of EGF-like modules, a large N-glyosylated spacer region followed by three repeated stretches of six cysteine residues with regular spacing, and one CUB domain at the C-terminus. Overexpression of human SCUBE2 protein, as for SCUBE1 and SCUBE3 [6,7], resulted in a secreted and membrane-tethered N-linked glycoprotein (Figures 2 and 3). Moreover, our mapping of the secretion and membrane-association domain to the spacer region and/or cysteine-rich repeats allowed us, for the first time, to assign one specific function to a structural domain of SCUBE2.
Of note, a fraction of the SCUBE2-FL or ty97 protein underwent limited proteolysis, producing a cleaved product of approx. 40 kDa released into the conditioned medium (Figure 2B). This finding is consistent with the presence of a unique insertion cassette of 30 amino acids between the EGF-like repeats 5 and 6, thus providing a potential furin cleavage site (Arg-Xaa-Lys-Arg, residues 276–279) only found in SCUBE2 (Figure 1). Interestingly, recent studies reported that active EGF-like repeat fragments could be liberated by matrix metalloproteases from two human extracellular matrix proteins, laminin γ2 chain and tenascin-C, to promote tumour cell migration and invasion through engaging the EGF receptor [31–33]. However, the exact identity of cellular protease(s) and the biological relevance of the proteolysis of SCUBE2 protein remains to be investigated further.
Our gain- and loss-of-function studies by ectopic overexpression and specific RNAi-mediated silencing experiments unravel a novel function for SCUBE2 in the SHH pathway by acting as a signalling component through interactions with SHH ligand and PTCH1 to promote its signal transduction (Figures 4 and 5). Together, our data suggest that the caveolin-1- or raft-associated SCUBE2 protein may concentrate the HH ligand within membrane raft microdomains and facilitate the presentation of HH ligand to the PTCH1 receptor in the responding cells.
In addition, our deletion analyses revealed that SCUBE2 binding to SHH or PTCH1 through the C-terminal CUB domain is necessary, but not sufficient, to enhance SHH signalling (Figure 5A). Moreover, SCUBE2 probably promotes signalling by binding SHH or PTCH1 through its C-terminal CUB domain in co-operation with interactions requiring other regions at the N-terminus, such as the EGF-like repeats or the spacer region (Figure 6). Although our data suggest a physical interaction between SCUBE2 and SHH or PTCH1 when overexpressed in HEK-293T cells or by an in vitro pull-down assay (Figure 4), whether such interactions indeed take place in vivo remains to be confirmed.
Our results not only provide potential biochemical and molecular explanations for the genetic interaction between SCUBE2 and HH observed in the zebrafish model system [1–3], but also are broadly in line with the results derived from the genetic analysis, which supports SCUBE2 acting as a permissive factor in promoting SHH signalling upstream of activation of SMO (the signalling component of HH receptor) (Figure 5C). However, we cannot exclude the possibility that a secreted extracellular form of SCUBE2 may also be involved in transporting and/or protecting the HH ligand from degradation in the extracellular environment. Alternatively, SCUBE2 could also participate in the process of releasing the HH ligand from the producing cells.
One important finding resulting from the present study is that SCUBE2 protein can be isolated from the buoyant caveolae or raft microdomains, and its interaction with calveolin-1 may represent a potential mechanism for recruitment of SCUBE2 into the specialized caveolin-1- or cholesterol-enriched microdomains. Because caveolin-1 is a major structural component of caveolae  and because many functions have been assigned to caveolae including endocytosis or transcytosis, cholesterol transport and homoeostasis, as well as both positive and negative regulation of Ras, Src, G-protein-coupled receptor and growth factor receptor signalling , further investigating whether SCUBE2 is also involved in the wide variety of diverse functions that has been ascribed to caveolin-1 and caveolae is of interest.
In summary, our findings that SCUBE2 is a novel component of the mammalian HH signalling pathway are of biological significance, because mouse Scube2 mRNA is expressed in the forebrain and the neural tube , where tightly regulated SHH activity is required for proper patterning and neural cell fate determination during early embryonic development . In addition, recent microarray gene expression profiling and immunohistochemiacl analyses revealed that the SCUBE2 transcript is associated with a number of human tumours such as breast tumours [36–38], where overreactive HH signalling has been implicated in breast cancer cell proliferation and breast cancer progression [39,40]. Further dissection of the molecular functions of SCUBE2 will provide a better understanding of its role in shaping and transducing SHH gradients and SHH-related human abnormalities.
Ming-Tzu Tsai and Chien-Jui Cheng performed the cloning and expression of SCUBE2, its biochemical interactions with SHH and caveolin-1. Yuh-Charn Lin demonstrated the direct interaction between SCUBE2 and SHH by in vitro pull-down assay. Chun-Chuan Chen, Ann-Ru Wu and Min-Tzu Wu conducted the Gli-luciferase reporter assay, Western blot analysis, SHH cell-based binding assay and biochemical fractionation of lipid rafts. Cheng-Chin Hsu and Ruey-Bing Yang conceived and supervised the overall project, designed biochemical and molecular biology experiments, edited and co-wrote the paper.
This work was supported by the Institute of Biomedical Sciences [grant number IBMS-CRC96-P01], Academia Sinica [grant number AS-97-FP-L16], and the National Science Council [grant numbers NSC 97-2752-B-006-003-PAE and NSC 97-2752-B-001-002-PAE to R.-B.Y and NSC 96-2320-B-038-027 to C.-J.C.). RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National Research Program for Genomic Medicine Grants of the National Science Council [grant numbers NSC 94-3112-B-001-003 and NSC 94-3112-B-001-018-Y].
We thank Ti-Yu Lin, Misty Lo and Cheng-Fen Tu for excellent technical assistance.
Abbreviations: AP, alkaline phosphatase; DMEM, Dulbecco's minimal essential medium; EGF, epidermal growth factor; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK, human embryonic kidney; HH, Hedgehog; PTCH1, Patched-1; RNAi, RNA interference; RT, reverse transcription; SCUBE, signal peptide, CUB domain, EGF-like protein 2; SCUBE-FL, full-length SCUBE; SHH, Sonic Hedgehog; shRNA, short hairpin RNA; SMO, Smoothened
- © The Authors Journal compilation © 2009 Biochemical Society