Fig 1.
Comparison of canine prominin-1 with its human and mouse orthologs.
(A) Membrane topology. Canine prominin-1 contains an extracellular N-terminal domain (EC1), five transmembrane segments (1–5) separating two small intracellular (IC1 and IC2), two large extracellular (EC2 and EC3) loops, and an intracellular C-terminal domain (IC3). The latter harbors a phosphorylation (P) site at tyrosine residue 829 for Src and Fyn tyrosine kinases. The asterisk stretch indicates a cysteine-rich region at the transition of the first transmembrane segment and IC1 domain. The EC2 and EC3 domains contain nine potential N-glycosylation sites (forks). The positions of the alternative exons 3 (purple), 11a (green), 19 (pink) and 22 (brown) found in the extracellular domains are indicated. (B) Genomic organization. The organization of human (h) and mouse (m) PROM1 genes is shown and compared to the canine equivalent (top panel). Vertical lines indicate exon boundaries and dark blue zones highlight the position of the transmembrane segments. Exon numbering begins with the one bearing the start codon. For simplicity, the details of the 5’-untranslated region (UTR) are not shown (see S1B Fig). Facultative exons included in the coding sequences are depicted in different colors and amino acid sequences of exons 3, 11a, 19 and 22 are indicated in parentheses (see also S2A Fig). The new canine prominin-1 splice variants (s22, s23) and two additional predicted ones (X1, X2) including their GenBank accession numbers are presented (bottom panel). The presence (+) or absence (−) of an exon and the consequential protein sequence length (number of amino acids) are indicated. T represents the stop codon within exon 27. (C) Amino acid identity. The percentage of amino acid identity of each individual structural domain of canine (c) prominin-1 with its corresponding counterparts in human and mouse orthologs is presented.
Table 1.
Studies on canine tissues and/or cell lines using anti-prominin-1 antibodies.
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.
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.
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.
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.
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.