Identification and Molecular Cloning of Canine Prominin-1 Splice Variants

Following in silico analysis and molecular cloning approaches (for the cloning strategy see Materials and Methods and S1A Fig), we identified and amplified distinct splice variants of canine prominin-1 from cDNA pools originating either from kidneys of adult beagles or MDCK cells (Fig 1 and S1 Fig; GenBank accession numbers KJ654317 and KR758755). The kidney was chosen as a mRNA source because prominin-1 has strong renal expression in rodents and humans [10, 12]. Predicted prominin-1 transcripts were also found in the NCBI database (GenBank entries: XM_845738, XM_005618555 and XM_005618556). Comparing these sequences with the corresponding prom1 gene sequence located on chromosome 3 (NW_003726054.1) using the Basic Local Alignment Search Tool (BLAST) program showed that introns are conserved in position and phase with human and mouse prom1 genes (Fig 1B and S1 Table). All exon/intron boundaries in the canine ortholog conform to the GT-AG rule (S1 Table).

The ORF showed that symmetric exon 3 –which encodes the 9-amino acid stretch PENVVLTLK in the extracellular N-terminal domain of prominin-1 –is alternatively spliced in the dog as previously reported for prominin-1 splice variants s1 and s2 in human and mouse [62, 63] (Fig 1A and 1B). We also highlighted one cDNA clone that lacks another exon (exon 22) in the third extracellular domain (Fig 1A and S2A Fig). Two other exons (11a and 19) are located in the second and third extracellular domains, respectively, and are predicted to be facultative (Fig 1B and S1 Table). Interestingly, the mini-exon 11a (residues ALP), which is located between exons 10 and 11 according to human (or mouse) gene structure, is also reported for amphibians and fish, but not yet in mammals [22, 25]. Strikingly, exon 27 in the dog harbors a stop codon generating a truncated cytoplasmic C-terminus that has not been found in any other species until now (Fig 1B and S2B Fig). This exon contains the entire 3’-untranslated region (UTR) and a polyadenylation consensus sequence (AAUAAA) 12 nucleotides upstream of a poly (A) tract (S2B Fig). Interestingly, analysis of the prom1 gene revealed the presence of a facultative exon 26a (SSVLGTWHFTL) between exons 24 and 27 (S2B Fig), which was found in mouse prominin-1.s3 to s6 variant and is predicted for human and rat (S2C Fig) [21]. The use of this exon creates a distinct cytoplasmic C-terminal end. Exons 25, 26b and 28 reported in mouse and/or human prom1 genes have not yet been identified in dog (Fig 1B and S1 Table).

Besides the ORF and in agreement with the existence of various 5’-UTR isoforms of human prominin-1 [64, 65], we amplified two alternative exons within the 5’-UTR (named A and B); a third one (C) is predicted (S1B Fig). The expression of different 5’UTRs in humans is tissue-specific and can vary with certain diseases (e.g., gliomas, colon cancers) (reviewed in Ref. [66]). In sum, these data suggest complex gene regulation of canine prominin-1 as reported for its counterparts in humans and mice. The functional significance of the alternatively spliced variants is unknown, but it indirectly suggests various interactions with other proteins—particularly by its alternative cytoplasmic C-terminal domain.

Following the nomenclature of prominin-1 splice variants [22, 23]–and with regard to the absence or presence of exon 3 –we named the new canine prominin-1 splice variants s22 and s23, respectively. They encode for 839 and 848 amino-acid proteins, respectively (Fig 1B).

Modest Amino Acid Conservation of Mammalian Prominin-1

A multiple sequence analysis of canine prominin-1 with human, mouse and rat orthologs revealed that the common characteristics of this pentaspan membrane glycoprotein are conserved (Fig 2, data not shown) [24]. For instance, six cysteine residues in the first and second extracellular loop are likely to form disulfide bridges and are found in all species (Fig 2, letter C in red). The unusual extra cysteine (residue 624) in human prominin-1 is absent in the canine sequence like in mouse and rat.

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Fig 2. Comparison of the amino acid sequence of canine, human and mouse prominin-1.

The canine prominin-1.s23 sequence determined in this study (GenBank Accession No. KR758755; top) was aligned with human (AF027208; middle) and mouse prominin-1.s2 (NM_001163577; bottom). Black and grey backgrounds indicate identical and similar amino acid residues, respectively; dashed line, putative signal peptide; solid blue lines, predicted transmembrane segments; solid red line, exon 3; asterisk stretch, cysteine-rich region; red C, conserved cysteine residue in extracellular domains; #, potential N-glycosylation site in canine prominin-1; yellow box, conserved lysine potentially involved in the interaction with HDAC6; green box, conserved Src/Fyn phosphorylation (P) site; and arrowhead, exon boundaries.

https://doi.org/10.1371/journal.pone.0164079.g002

The cysteine-rich sequence at the transition of the first transmembrane domain and the first cytoplasmic loop is also present in canine prominin-1 (Fig 2, asterisks). The lysine 138 within the cysteine-rich domain, which is implicated in the interaction with histone deacetylase 6 (HDAC6), is conserved (Fig 2, yellow box) [67]. This holds true for the tyrosine 829 that can be phosphorylated by Src and Fyn tyrosine kinases in human prominin-1 (Figs 1A and 2, green box, P) [68]. The latter posttranslational modification can promote the interaction of prominin-1 with the regulatory subunit (p85) of phosphoinositide 3-kinase (PI3K) leading to the activation of PI3K/Akt pathway [69]. Nine N-glycosylation sites are found in the extracellular domains of canine prominin-1. Only two of them are conserved in position with human and mouse proteins (Fig 2, symbol #).

The pairwise sequence comparison revealed that canine prominin-1 exhibits an overall amino acid identity of 66% and 57% with its human and mouse orthologs, respectively, while the human and mouse sequences show 61% identity. The similarity of canine prominin-1 reaches 74% and 66% with human and mouse proteins, respectively. Although the two small intracytoplasmic loops display a significant amino acid identity between mammalian prominin-1 (79 to 95%), the three large extracellular domains are modestly conserved (Fig 1C). Only 60% of the residues exposed to the extracellular milieu are conserved between the canine and human proteins, and this number decreases to 45–55% in the murine sequence. The cytoplasmic C-terminal domain of canine prominin-1 is significantly different between human and mouse due to the presence of a stop codon in exon 27 (Figs 1B and 2).

Given the limited amino acid conservation between the canine prominin-1 and various mammalian orthologs, the cross-species reactivity of antibodies directed against human or mouse prominin-1 with the canine protein is questionable—particularly when they are directed against the large extracellular loops or the cytoplasmic C-terminal end.

Canine Prominin-1 Is Targeted to the Apical Plasma Membrane of Polarized Epithelial Cells

To further characterize the canine prominin-1, we ectopically expressed it as a GFP-fusion protein in MDCK cells. We expressed the canine prominin-1.s23 variant because it has the same extracellular domains as the human prominin-1, which is recognized by commercial and homemade mAbs (see below). The GFP allows us to monitor its expression in the absence of specific antibodies. We have previously demonstrated that the addition of GFP in-frame with the cytoplasmic C-terminal domain of human or mouse prominin-1 did not interfere with their membrane topology nor their proper intracellular trafficking and targeting to a specific subdomain of polarized cells (see also below) [14, 70].

To determine the distribution of canine prominin-1-GFP in polarized epithelial cells, we grew MDCK cells for 6 days post-confluence. Confocal laser scanning microscopy analysis revealed the presence of the fusion proteins at the apical domain of cells with a punctate staining pattern typical for microvilli (Fig 3A–3C mv). The recombinant protein was also found in primary cilia, which are highlighted by acetylated tubulin labeling (Fig 3A–3C, pc; S1 Video). The single x-z (Fig 3B) and x-y sections (Fig 3C) indicate a fluorescent signal in cytoplasmic vesicles consistent with the intracellular transport of prominin-1-GFP en route to the apical plasma membrane (asterisks). No significant GFP-fluorescence was observed at the basolateral domain (Fig 3B and 3C, S1 Video). The same distribution pattern of human prominin-1-GFP fusion protein was observed upon its overexpression in MDCK cells (Fig 3A and 3C). Thus, canine prominin-1 is targeted to the apical plasma membrane of polarized epithelial cells and concentrated in plasmalemmal protrusions found therein as previously reported for human and mouse prominin-1 [11, 12, 14, 71–73].

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Fig 3. Canine prominin-1-GFP is found in microvilli and the primary cilium at the apical domain of polarized epithelial cells.

(A-C) Stably transfected MDCK cells expressing either canine (c) or human (h) prominin-1-GFP (Prom1-GFP, green) were immunolabeled for acetylated α-tubulin (ac Tub, red) and their nuclei were counterstained with DAPI (blue). 3-dimensional reconstruction of 32 optical x-y sections (0.6 μm-slices) (A) and a single x-z section (B) are shown. The three single x-y sections (1–3) displayed in (C) are located as illustrated in the cartoon above. They reveal the presence of prominin-1-GFP at the apical plasma membrane with fluorescent signals characteristic of microvilli (mv) and the primary cilium (pc, arrow; see also S1 Video) as well as in intracellular structures such as the endoplasmic reticulum (ER, asterisk). Tj, tight junction. Scale bars, 5 μm.

https://doi.org/10.1371/journal.pone.0164079.g003

Commercial Monoclonal Antibodies Do Not Recognize Canine Prominin-1 by Immunocytochemistry and Flow Cytometry

To evaluate if commercial antibodies directed against human and mouse prominin-1 can cross-react with the canine protein as assumed in several studies (Table 1), we established stably transfected MDCK cells expressing human and mouse prominin-1 as positive controls, in addition to canine prominin-1-GFP⁺ cells. We chose splice variants that are recognized by mAbs directed against human (i.e. clones AC133, AC141 and 293C3) and mouse (clone 13A4) prominin-1 [12, 37]. The CD133/1 (recognized by AC133 antibody) and CD133/2 (recognized by AC141 and 293C3 antibodies) epitopes are found in the second extracellular loop of human prominin-1 [74, 75], while the same domain in mouse prominin-1 contains the 13A4 epitope [8]. The structural domains of both extracellular loops, in terms of exons, are conserved within the dog prominin-1.

The cell surface immunostaining of untransfected MDCK cells with the above antibodies revealed that none of them detected canine prominin-1 (Fig 4A). More strikingly, the overexpressed canine prominin-1-GFP was not recognized by any tested mAb (Fig 4A). In contrast, the antibodies could detect either human or mouse prominin-1 (Fig 4A). To rule out that canine prominin-1-GFP did not reach the cell surface but instead localized to vesicular structures underneath the plasma membrane, we repeated the staining upon cell permeabilization via a low concentration of saponin. Again, the anti-prominin-1 antibodies failed to detect the endogenous and ectopically expressed canine prominin-1 (Fig 4B).

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Fig 4. Commercially available anti-human and anti-mouse antibodies fail to detect canine prominin-1 by cytochemistry and flow cytometry.

(A-C) The MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed either by immunocytochemistry (A, B) or flow cytometry (C) using mAbs AC133, AC141, 293C3 and 13A4. For immunocytochemistry, cells were either cell surface labeled in the cold (A) or permeabilized with saponin after fixation (B). As negative controls, only the secondary antibody was used, and cells were counterstained with DAPI (A, B). Scale bars, 30 μm. For flow cytometry, cells were directly incubated with fluorochrome (PE or APC)-coupled mAbs as indicated. Cells expressing prominin-1-GFP were examined using the FITC channel. The vertical dashed lines indicate the cut-off for cells positive for a given prominin-1. These were established using unstained cells or those labeled with the irrelevant anti-human CD34 antibody conjugated to the appropriate fluorochrome (S4 Fig). The color code indicates the corresponding cell line used, and the percentage of positive cells is specified in each panel.

https://doi.org/10.1371/journal.pone.0164079.g004

We next wondered if the addition of GFP at the cytoplasmic C-terminal domain of canine prominin-1 somehow influences its tertiary structure—particularly the second extracellular loop—in a way that impedes the epitope accessibility. To answer this question, we repeated the immunocytochemistry using the human prominin-1-GFP fusion protein where the GFP is also added at the C-terminal end. In contrast to canine prominin-1-GFP, we could detect the fusion protein with all mAbs (i.e. AC133, AC141 and 293C3) directed against human prominin-1 (S3A Fig). This indicated that the protein tag did not influence the accessibility of the corresponding epitopes. Again, the mAb 13A4 did not recognize the human prominin-1 (S3A Fig). The lack of immunodetection of canine prominin-1 can also result from other molecular constrains—notably the incorporation of prominin-1 in ganglioside GM₁-enriched membrane microdomains that occurs when epithelial cells are grown as a polarized monolayer [10, 73, 76] (reviewed in Ref. [77]). Thus, we re-analyzed MDCK cells with flow cytometry after substrate detachment. We used antibodies that were directly coupled to a fluorochrome. None of these antibodies cross-reacted with endogenous prominin-1 or canine prominin-1-GFP, although the anti-human and anti-mouse prominin-1 antibodies highlighted their respective antigen (Fig 4C). No positive cells were observed without primary antibody or with an irrelevant antibody, i.e. anti-human CD34 antibody (S4 Fig).

Commercial Monoclonal Antibodies Do Not Recognize the Canine Prominin-1 by Immunoblotting

We investigated whether commercial antibodies can detect canine prominin-1 by immunoblotting. Proteins in detergent lysates prepared from the established MDCK cell lines were separated by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) under reducing condition (i.e. in presence of β-mercaptoethanol) and analyzed by blotting. As expected from the immunocytochemistry and flow cytometry, none of the antibodies directed against human and mouse prominin-1 detected the endogenous prominin-1 of MDCK cells or its overexpressed GFP fusion protein (Fig 5A). The latter can be observed using an anti-GFP antibody and shows the expected higher molecular weight. The two-band pattern of recombinant prominin-1 (canine, human and mouse proteins) is consistent with its overexpression as demonstrated in earlier reports [12, 21, 71]. The immunoreactive band with an apparent molecular mass of ≈115–120 kDa (or ≈160–170 kDa in the case of GFP fusion protein) represents proteins at the plasma membrane (Fig 5A, arrow). The ≈100–105 kDa (≈150 kDa for prominin-1-GFP) forms are found in the endoplasmic reticulum and/or an early Golgi compartment (Fig 5A, arrowhead). The antibodies also failed to detect the canine prominin-1 under non-reducing conditions (Fig 5B). The mAbs AC133 and 293C3 detected a faint band at ≈190 kDa (Fig 5B, asterisk) suggesting the formation of dimers or multimers.

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Fig 5. The commercial anti-human and anti-mouse antibodies fail to detect canine prominin-1 by immunoblotting.

(A-C) Detergent lysates prepared from MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed by SDS-PAGE under reducing (A, C) and non-reducing (B) conditions and immunoblotting using mAbs AC133, AC141, 293C3, and 13A4 or polyclonal antibody against GFP. β-actin and α-tubulin were used as loading controls. As negative controls, only the secondary antibody (as indicated) was used (C). (D) Lysates from canine prominin-1-GFP transfected cells were incubated with (+) or without (–) PNGase F prior to immunoblotting with anti-GFP antibody or others (as indicated). The arrow and open arrowhead indicate the plasma membrane-associated form and endoplasmic reticulum-associated form of prominin-1, respectively. Asterisk indicates potential disulfide-bridged prominin-1 dimers or multimers, and the black arrowhead shows deglycosylated prominin-1. Molecular mass markers (kDa) are indicated. The original and uncropped blots are presented in S5 Fig.

https://doi.org/10.1371/journal.pone.0164079.g005

Given that we were monitoring the GFP-fusion protein for canine prominin-1, we also performed the immunoblotting of lysates from MDCK cells expressing human prominin-1-GFP. Again, anti-human prominin-1 antibodies recognized the human, but not the canine, fusion protein (S3B and S3C Fig). The expression levels of human and canine GFP-fusion proteins were comparable (S3D Fig).

Because of the debate about the role of glycosylation in the recognition of certain human prominin-1 epitopes, notably CD133/1 (reviewed in Ref. [78]), we decided to remove the N-glycans prior to immunoblotting. Detergent extracts from MDCK cells expressing canine prominin-1-GFP were treated with PNGase F and analyzed by SDS-PAGE under reducing conditions. Upon detection with an anti-GFP antibody, the recombinant fusion protein is glycosylated as observed by the shift of the molecular mass from ≈150/170 to 115 kDa (Fig 5D, black arrowhead). This is consistent with the presence of nine N-glycosylation sites (Fig 2). However, the removal of sugar moieties did not unmask any detectable epitope so again none of the anti-human or mouse prominin-1 antibodies could recognize the canine prominin-1 (Fig 5D).

Homemade Antibodies Do Not Recognize the Canine Prominin-1

Our laboratory has developed a mouse mAb and rabbit antiserum named 80B258 and αhE2, respectively, against human prominin-1 [10, 11]. They were generated using a fragment of its first extracellular loop corresponding to amino acid residues 240–388 of splice variant s2 as the antigen (Fig 2). This region exhibits 59% amino acid identity and 68% similarity between human and canine prominin-1. Only 49% amino acid identity is observed with the mouse ortholog. Both mAb 80B258 and antiserum αhE2 failed to recognize the endogenous prominin-1 as well as the ectopically expressed canine prominin-1-GFP in MDCK cells by immunocytochemistry and immunoblotting (Fig 6A and 6B, respectively). They recognized human prominin-1 or its GFP version, but not mouse prominin-1 (Fig 6 and S3B Fig), in agreement with a previous report [11].

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Fig 6. Homemade mouse monoclonal antibody 80B258 and rabbit antiserum αhE2 fail to detect canine prominin-1.

(A, B) MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed either by immunocytochemistry (A) or immunoblotting under reducing conditions (B) using mouse mAb 80B258 or rabbit antiserum αhE2. As a negative control, only the secondary antibody (as indicated) was used (A, B). For immunocytochemistry, cells were counterstained with DAPI (A). For immunoblotting, β-actin was used as loading control (black arrowhead). The arrow indicates the plasma membrane-associated form of prominin-1, and the open arrowhead indicates its endoplasmic reticulum-associated form. The original and uncropped blots are presented in in S5 Fig. Scale bar, 30 μm.

https://doi.org/10.1371/journal.pone.0164079.g006

Molecular Cloning of Canine Prominin-1

The nucleotide and protein databases at the National Center for Biotechnology Information (NCBI) were searched by BLAST network services [91] using the human and mouse prominin-1 nucleotide sequences (GenBank accession numbers AF027208 and F026269, respectively) as probes. We obtained a predicted canine prominin-1 sequence (transcript variant X1, #XM_845738.4) derived from the whole genome shotgun sequence of chromosome 3 of canis lupus familiaris breed boxer (#NW_003726054.1). Based on this information, we designed various oligonucleotide primers (S2 Table, primer Nr. 1–5) to selectively amplify the canine prominin-1 by polymerase chain reaction (PCR). As a template, we prepared a cDNA pool from total RNA of MDCK cells (strain II, obtained from American Type Culture Collection, #ATCC^®CCL-34^™) using MultiScribe^™ reverse transcriptase (#4368814, Applied Biosystems, Thermo Fisher Scientific). As a result, a 1.9-kb fragment of canine prominin-l was amplified (see S1A Fig). However, it did not contain the 5’- and the 3’-ends. The missing extremities were amplified by 5’- and 3’-rapid amplification of cDNA ends (RACE) using SMARTer^™ RACE cDNA amplification kit according to the manufacturer’s instructions (#634858, Takara Bio Europe/Clontech, France). All resulting PCR DNA fragments were gel extracted (QIAquick Gel Extraction Kit, #28704, Qiagen GmbH, Helden, Germany) and subcloned into pCR^™4-TOPO^® TA vector (Invitrogen). The identity of the inserts was determined by sequencing. This strategy identified three splice variants affecting the open reading frame of canine prominin-1 and two affecting its 5’UTR (S1 and S2 Figs).

The three overlapping cDNA fragments were combined using oligonucleotide primers carrying different specific restrictions sites (S2 Table, Nr. 8–13) for amplification. Briefly, the 5’-RACE cDNA fragment was subcloned into the pEGFP (enhanced green fluorescent protein)-N1 vector (Takara Bio Europe/Clontech) opened with XhoI and BamHI enzymes. The 1.9-kb fragment was ligated to the 5’-end after digestion with the SbfI and BamHI enzymes. The 3’-RACE cDNA fragment was then inserted in the same way using the introduced SacI and BamHI restriction sites. All restriction sites introduced for subcloning were removed using QuikChange^® II Site-Directed Mutagenesis Kit according to the manufacturer’s instructions (#200523, Agilent Technologies) with the oligonucleotide primers (Nr. 16–19) described in S2 Table. Note that the ORF of the prominin-1.s23 sequences are not in-frame with the GFP.

We also used PCR to obtain the canine prominin-1.s22 cDNA sequence from adult beagle dog kidney cells (dog kidney full length tissue cDNA, #DD-901, Zyagen, San Diego, CA) using oligonucleotide primers 6 and 7 (S2 Table). The resulting PCR DNA product was subcloned into pCR^™4-TOPO^® TA vector.

All DNA fragments and the final sequences were verified by sequencing to confirm the absence of any reading mistake introduced by the DNA polymerase. The sequencing was performed at the Sequencing Facility of the Max Planck Institute of Molecular Cell Biology and Genetics (Dresden, Germany). The resulting sequences were deposited in GenBank database under the following accession number KJ654317.1 and KR758755.1; they are also presented in S1 and S2 Figs.

Expression Plasmid Construction

The eukaryotic expression plasmid containing canine prominin-1.s23 sequence in-frame with GFP was generated by removing the terminal codon of prominin-1 as well as one adenine residue using QuikChange^® II Site-Directed Mutagenesis Kit with the oligonucleotide primers 14 and 15 (S2 Table). A linker of 11 amino acids is located between the prominin-1.s23 and the GFP sequence. The eukaryotic expression plasmids containing either mouse prominin-1.s1, human prominin-1.s2 or human prominin-1.s2-GFP have been described previously [12, 70, 72].

Protein Sequence Analysis

The amino acid sequence deduced from canine prominin-1 cDNAs was compared with its mouse and human orthologs using CLUSTAL OMEGA [92, 93]. Pairwise sequence identity and similarity were analyzed using Sequence Identity And Similarity (SIAS) with BLOSUM62 matrix (gap open and gap extend penalties at 10 and 0.5, respectively) [94] on a Spanish server (http://imed.med.ucm.es/Tools/sias.html). The signal peptide and transmembrane domains were determined using the Consensus Constrained TOPology prediction web server (http://cctop.enzim.ttk.mta.hu) [95]. The Color Align Conservation program from Sequence Manipulation Suite (SMS; www.bioinformatics.org) was used to illustrate the output of the sequence alignment programs [96].

Cell Culture and Transfection

MDCK cells (see above) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 1x, 1g/l D-Glucose, L-Glutamine, Pyruvate, GIBCO) containing 10% fetal calf serum (FCS, PAA, GE Healthcare), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO), 10 mM HEPES and MEM Non Essential Amino Acids (1x, PAA) at 37°C in a humidified 5% CO₂ atmosphere as described previously [71]. They were stably transfected with eukaryotic expression plasmids encoding either canine prominin-1.s23-GFP (see above), human prominin-1.s2, human prominin-1.s2-GFP [70, 72, 97] or mouse prominin-1.s1 [12] by electroporation using Amaxa^™ Nucleofector^™ Kit L according to the manufacturer’s instructions (#VCA-1005, Lonza, Cologne, Germany). Cells expressing the neomycin resistance gene were selected by introducing 500 μg/ml of geneticin (Thermo Fisher Scientific) into the culture medium and further expanded in a medium containing only 250 μg/ml of selective agent. To enhance the expression of the transgene, 10 mM sodium butyrate was added to the culture medium for 16 h prior to the analyses [71]. Under these conditions, 50–90% of neomycin-resistant cells expressed the recombinant prominin-1.

Immunofluorescence and Confocal Microscopy

Cell surface labeling—Wild type and transfected MDCK cells were grown on fibronectin (BD Biosciences)-coated glass coverslips or in eight-well chambers (μ-Slide 8 Well, Ibidi, #80826) until day 6 post-confluence. Cells were rinsed with Ca/Mg buffer (phosphate-buffered saline containing 1 mM CaCl₂ and 0.5 mM MgCl₂) and incubated in blocking buffer (Ca/Mg buffer containing 0.2% gelatin) for 30 min at 4°C. Cells were incubated with either mouse mAb AC133 (epitope CD133/1; 1:50, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), AC141 (epitope CD133/2; 1:50, Miltenyi Biotec), 293C3 (CD133/2, 1:50, Miltenyi Biotec) or rat mAb 13A4 (1:100, eBioscience) diluted in blocking buffer for 1 h at 4°C. Cells were washed with PBS and then fixed in 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature. Coverslips were then rinsed in PBS containing 50 mM NH₄Cl and incubated in this same solution for 10 min.

Intracellular labeling—PFA-fixed cells were permeabilized and blocked with PBS containing 0.2% gelatin and 0.2% saponin (Panreac Applichem, Darmstadt, Germany) for 30 min at room temperature prior to the incubation with anti-prominin-1 antibodies for 30 min at room temperature. To visualize the primary cilium cells were labeled with a mouse mAb anti-acetylated α-tubulin (1:1000, clone 6-11B-1, #T6793, Sigma-Aldrich) followed by Alexa546-conjugated goat anti-mouse IgG_2b (1:600, Molecular Probes—Thermo Fisher Scientific) for 30 min in permeabilization buffer. For labeling with homemade antibodies, PFA-fixed cells were incubated in SDS buffer (0.005% SDS and 0.2% gelatin in PBS) for 30 min at room temperature and washed with PBS containing 0.2% gelatin for 10 min. Cells were labeled with mouse mAb 80B258 (10 μg/ml, [11]) or rabbit antiserum αhE2 (1:500, [10]) diluted in PBS containing 0.2% gelatin for 30 min at room temperature.

In all cases, Alexa555/546-conjugated goat/donkey anti-mouse, anti-rabbit or anti-rat IgG antibodies (1:500, Life Technologies) were used to detect anti-prominin-1 antibodies. Nuclei were counterstained with 4’-6-diamidino-2-phenylindole (DAPI, 1 μg/ml, Sigma-Aldrich). Samples were washed three times in PBS containing 0.2% gelatin, once in PBS, once in H₂O and mounted in Mowiol 4.88 (Merck, Darmstadt, Germany). Cells were observed with a Zeiss LSM 700 confocal laser scanning microscope. The images in Fig 3A and S1 Video were prepared from data files using Volocity 3D Image Analysis Software (PerkinElmer) and all others were processed with Fiji [98].

Flow Cytometry

Wild type and transfected MDCK cells were harvested via treatment with 0.05% trypsin/0.5 mM EDTA solution (Life Technologies) for 15 min at 37°C. Cells were centrifuged, and the pellet was resuspended in PBS containing 0.2% gelatin and 5 mM EDTA (VWR International, Radnor, Pennsylvania, USA). Cell suspensions (2 x 10⁵ cells in 100 μl) were incubated for 30 min at 4°C with fluorochrome-conjugated antibodies: mouse mAb AC133-phycoerythrin (PE) (1:10, Miltenyi Biotec), AC141-PE (1:10, Miltenyi Biotec), 293C3-allophycocyanin (APC) (1:10, Miltenyi Biotec) and CD34-PE or CD34-APC (1:10, BD Bioscience) or rat mAb 13A4-PE (1:10, eBioscience). Cells expressing prominin-1-GFP were examined using the fluorescein isothiocyanate (FITC) channel. Cells were washed with PBS containing 5 mM EDTA. After centrifugation, cells were resuspended and incubated for 10 min in 4% PFA. After washing with PBS containing 5 mM EDTA, 10,000 events were acquired on a FACSCanto II System (BD Bioscience). Instrument settings and gating strategies were established using unstained or CD34-labeled MDCK cells.

Detergent Cell Extracts and Endoglycosidase Digestion

Wild type and transfected MDCK cells (2 x 10⁶) were grown in 6-well plates until 3 days post-confluence. Cells were harvested by trypsinization and centrifuged for 5 min at 300 x g. Cell pellets were lysed in solubilisation buffer (1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecylsulfate, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0) supplemented with complete protease inhibitor (Roche Molecular Biochemicals) for 30 min at 4°C. Lysates were centrifuged at 4°C (10 min, 10,000 x g) and the resulting supernatants were either used for endoglycosidase digestion or directly mixed with Laemmli sample buffer (with or without reducing agent; β-mercaptoethanol) prior to analysis with SDS-PAGE. For endoglycosidase digestion, the detergent cell extracts from MDCK cells expressing the canine prominin-1-GFP were incubated for 6 h at 37°C (or room temperature) in the absence or presence of 1 unit PNGase F (peptide-N-glycosidase F; #11 365 177 001, Roche Diagnostics GmbH).

Immunoblotting

Cell detergent extracts were prepared from wild type or transfected MDCK cells (corresponding to one-tenth of a unit of 6-well plate) and separated by SDS-PAGE (7.5%) under reducing or non-reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes (pore size: 0.45 μm; Millipore Corp, Belford, MA) using a semi-dry transfer cell system (Cti, Idstein, Germany). After transfer, membranes were incubated in blocking buffer (PBS containing 0.3% Tween 20 and 5% milk powder) overnight at 4°C. Incubation with primary antibodies against prominin-1; mouse mAb AC133 (1:100), AC141 (1:100), 293C3 (1:100), 80B258 (1:1500) or rat mAb 13A4 (1:500), and rabbit antiserum αhE2 (1:1500) was performed for 1 h at room temperature. The GFP fusion protein was detected using a rabbit anti-GFP polyclonal antibody (1:2000, #210-PS-1GFP, ImmunoKontact). As a loading control, mouse mAb AC-74 against β-actin (1:10,000, Sigma-Aldrich) or DM1A against α-tubulin (1:10,000, Sigma-Aldrich) were used. All antibodies were diluted in blocking buffer. Antigen-antibody complexes were detected using appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Inc.) and visualized with enhanced chemiluminescence reagents (ECL system, GE Healthcare). Membranes were exposed to films (GE Healthcare).

Statistical Analysis

Statistical analysis was performed using unpaired two-tailed student’s t-test. Differences were regarded as not significant if the calculated p-value was ≥ 0.05.