Cell Tissue Res, DOI 10.1007/s00441-016-2415-9

Identification of a shootin1 isoform expressed in peripheral tissues
Yasuna Higashiguchi1, Kazuhiro Katsuta1, Takunori Minegishi1, Shigenobu Yonemura2,
Akihiro Urasaki1, Naoyuki Inagaki1
1

Laboratory of Systems Neurobiology and Medicine, Graduate School of Biological Sciences,

Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
2

Department of Cell Biology, Institute of Biomedical Sciences, Tokushima University Graduate

School, Kuramoto-cho, Tokushima 770-8504, Japan
Email: Naoyuki Inagaki, ninagaki@bs.naist.jp

Abstract
Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon
outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates themechanical coupling between F-actin retrograde flow and cell adhesions, as a clutch molecule,thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform ofshootin1 which is expressed not only in the brain but also in peripheral tissues. We renamed thebrain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot andimmunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is
distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas,kidney and skin. Interestingly, shootin1b immunoreactivity was detected widely in epithelial cells
that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin andcortactin at cell-cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These
results suggest that shootin1b may function in various peripheral tissues including epithelial cells.
Keywords: Shootin1b, Clutch molecule, Epithelial cell, Dendritic cell1

Introduction
The linkage between the actin cytoskeleton and cell adhesions plays essential roles in cell-cellinteraction, cellular morphogenesis and migration (Suter and Forscher, 2000; Le Clainche andCarlier, 2008; Yonemura, 2011; Brieher and Yap, 2013). In motile cells, actin filaments
polymerize at the leading edge and disassemble proximally (Pollard and Borisy, 2003; Le
Clainche and Carlier, 2008), which, in conjunction with myosin II activity, induces retrograde
flow of actin filaments (Wang, 1985; Forscher and Smith, 1988; Katoh, et al., 1999; Mallavarapu
and Mitchison, 1999). Mechanical coupling between actin filament flow and cell adhesions by“clutch” molecules is thought to play key roles in producing force for axon outgrowth and cell
migration (Mitchison and Kirschner; 1988, Suter and Forscher, 2000; Thievessen, et al., 2013;
Toriyama, et al., 2013; Garcia, et al., 2015).
Shootin1 is a recently identified brain-specific clutch molecule (Toriyama, et al., 2006;
Shimada, et al., 2008) that is involved in axon outgrowth and neuronal polarity formation(Toriyama, et al., 2010; Inagaki, et al., 2011; Sapir, et al., 2013). In cultured hippocampal neurons,
shootin1 couples mechanically the actin filament retrograde flow and the cell adhesions at theleading edge of axonal growth cones, through the actin-binding protein cortactin (Weed andParsons, 2001; MacGrath and Koleske, 2012) and the cell adhesion molecule L1-CAM(Kamiguchi, et al., 1998), and produces force for axon outgrowth (Kubo, et al., 2015). This
coupling is positively regulated by the chemoattractant netrin-1 through PAK1-mediated shootin1
phosphorylation (Toriyama, et al., 2013; Kubo, et al., 2015). A recent report also demonstrates
that shootin1-mediated cytoskeletal-adhesion coupling contributes to an intracellular transport ofactin and actin-associated proteins along axons (Katsuno, et al., 2015).
In this study, we report a novel splicing isoform of shootin1, shootin1b, which isexpressed not only in the brain but also in peripheral tissues. To provide insights into the functionsof shootin1b, we examined its tissue localization using an antibody which specifically recognizes
shootin1b. Our data show that shootin1b is expressed in various cells including epithelial cells
and dendritic cells in peripheral tissues.

2Materials and Methods
All relevant aspects of the experimental procedures were approved by the Institutional Animal
Care and Use Committee of Nara Institute of Science and Technology.
Cloning of shootin1b
Full-length cDNA encoding rat shootin1b was amplified by PCR from a rat brain cDNA library(ClontechLaboratories)

with

the

CCGCTCGAGATGAACAGCTCGGACGAGGAGAAG
CCGCTCGAGTCAGCAGTTAGAACTGTCCGTCTC.

To

produceprimers

5’-

and

5’-

a

shootin1b-specificantibody, cDNA encoding the C-terminal region (amino acid residues 454-633) of rat shootin1b
was amplified by PCR with the primers 5’-CGGGATCCGGGACACTTAACAAACCCAC and5’-CCGCTCGAGTCAGCAGTTAGAACTGTCCGTCTC. The cDNAs were then subcloned into
the pGEX vector (GE Healthcare).
Protein and antibody preparation
Protein and antibody preparation were performed as described (Toriyama, et al., 2006). In brief,
recombinant full-length and C-terminal regions of shootin1b were expressed in Escherichia coli
as GST fusion proteins and purified on a glutathione-Sepharose column (GE Healthcare), after
which GST was removed by PreScission protease (GE Healthcare). A rabbit polyclonal antishootin1b antibody was raised against the recombinant C-terminal region of shootin1b andaffinity-purified before use.
Immunoblot
Immunoblotting was performed as described previously (Inagaki, et al., 1997). Adult (2-5 months)
and embryonic day (E) 18.5 mouse tissues were diced into small pieces in RIPA lysis buffer (50mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium
deoxycholate, 2 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 10 mM NaF and 1mMDTT) and sonicated. After a 15-min incubation on ice, protein extracts were centrifuged at
17,500g for 15 min at 4°C. After mixing with SDS loading buffer and incubating at 95°C for 5min, the protein extracts and purified proteins were electrophoresed on 8% polyacrylamide gelsand transferred to PVDF membranes. After blocking with blocking buffer (0.05% Tween-20, 3%
non-fat dried milk in Tris-buffered saline) for 1 h at room temperature, the membranes wereincubated with anti-shootin1 antibody (1:200 dilution in blocking buffer), anti-shootin1b antibody(1:20,000) or anti-actin antibody (1:5,000) overnight at 4°C. After washing, membranes wereincubated with horseradish peroxidase (HRP)-linked anti-rabbit antibody (1:2,000) or HRPlinked anti-mouse antibody (1:5,000) for 1 h at room temperature. Immunoreactive bands were3

visualized by the ECL Western Blotting Detection system (GE Healthcare).
Cell culture
EpH4 cells from mouse mammary glands (Reichmann, et al., 1989) (provided by Dr. E. Reichman,
University Children’s Hospital, Zurich, Switzerland) were cultured at 37°C in a humid
atmosphere of 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium (Sigma)
supplemented with 10% FBS (Invitrogen).
Immunohistochemistry, immunocytochemistry and microscopy
Immunohistochemistry and immunocytochemistry were performed as described (Inagaki, et al.,
1988; Inagaki, et al., 1997), with slight modifications. Briefly, mouse tissues were dissected andtrimmed to allow efficient penetration of the fixative. After fixation by 4% paraformaldehyde(PFA), tissues were dehydrated with 30% sucrose and embedded in Tissue-Tek O.C.T. Compound(Sakura) and cut into 8-μm sections on a Cryostat. The sections were washed and blocked withblocking buffer (10% normal goat serum or 10% Fetal bovine serum in phosphate buffer with
0.3% Triton X-100) for 2 h at room temperature. Samples were incubated with anti-shootin1b(1:20,000 dilution in blocking buffer), anti-TFF2 (1:200), anti-Mist1 (1:200), anti-cytokeratin-19(1:50), anti-albumin (1:200), anti-amylase (1:200), anti-E-cadherin (1:1,000), anti-cortactin(1:100) or anti-CD11c (1:100) overnight at 4°C, and then incubated with Alexa Fluor 488conjugated anti-Rabbit IgG (H+L) (1:1,000), Alexa Fluor 594-conjugated anti-Mouse IgG (H+L)(1:1,000) or Alexa Fluor 594-conjugated anti-Armenian hamster IgG (H+L) (1:500) overnight at
4°C. After washing, cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). For
preparation of the data in Fig. S3, two serial sections of skin and forestomach were
immunostained: one section stained with anti-shootin1b antibody and the other stained without
the antibody. Their photographs were taken at the same exposure time.
EpH4 cells were fixed with 1% formaldehyde in phosphate buffered saline (PBS) for 15
min at room temperature. After washing with PBS, the cells were treated with 0.2% Triton X-100
in PBS for 10 min at room temperature and blocked with blocking buffer (10% fetal bovine serum
in PBS) for 1 h at room temperature. They were then incubated with anti-shootin1b (1:10,000
dilution in blocking buffer) together with anti-E-cadherin (1:200) or anti-cortactin (1:100)
overnight at 4°C, and incubated with Alexa Fluor 488-conjugated anti-Rabbit IgG (H+L)(1:1,000) and Alexa Fluor 594-conjugated anti-Mouse IgG (H+L) (1:1,000) for 1 h at room
temperature.
Fluorescence images were acquired using a confocal microscope (LSM 700 or LSM
710; Carl Zeiss) equipped with a EC Plan-Neofluar 10x 0.30 M27, a plan-Apochromat 20x
0.8 M27, a plan-Apochromat 40x 1.3 oil DIC M27 and a plan-Apochromat 63x 1.40 oil M27
4objectives (Carl Zeiss) and imaging software (ZEN2009; Carl Zeiss). The acquired images were
processed using Adobe Photoshop.
RT-PCR
Total RNA was extracted from the cornified layer and whole skin of P5 mouse by using TRIzol(Thermo Fisher Scientific) according to manufacturer’s instructions, and then treated with RQ1RNase-free DNase (Promega). Fifty ng of total RNA was used for cDNA synthesis using M-MLV
Reverse Transcriptase (Promega) and Random Primer (Takara). PCR was performed with thecDNA as a template and ExTaq HS (Takara). Primers used for RT-PCR are as follows: 5’ATAGCAGTAGTCCTACCGGGATATT-3’(shootin1b

forward),5’-

TTTTCTCCTGTATCCACCTTTACTG-3’(shootin1b

reverse),

5’-

and

5’-

TGTTACCAACTGGGACGACA-3’(-actin

forward),GGGGTGTTGAAGGTCTCAAA-3’ (-actin reverse).
Materials
C57BL/6 mice were obtained from SLC Japan and CLEA Japan. Preparations of shootin1a andthe antibody against shootin1 were described elsewhere (Toriyama, et al., 2006). DAPI was
obtained from Roche Diagnostics. Monoclonal antibodies against CD11c (N418, Armenian
hamster), TFF2 (GE16C, mouse) and Mist1 (6E8/A12/C11P1, mouse) were obtained from Abcam.
Monoclonal antibodies against amylase (G-10, mouse), cortactin (4F11, mouse) and E-cadherin(mouse) were obtained from Santa Cruz Biotech, Millipore and BD Transduction Laboratories,
respectively. Polyclonal antibodies against cytokeratin-19 (N-13, goat) and albumin (goat) wereobtained from Santa Cruz Biotech and Bethyl laboratories, respectively. HRP-linked anti-rabbit
IgG, Alexa Fluor 488-conjugated anti-Rabbit IgG (H+L), Alexa Fluor 594-conjugated anti-Rabbit
IgG (H+L), Alexa Fluor 594-conjugated anti-Mouse IgG (H+L), Alexa Fluor 594-conjugated antiArmenian hamster IgG (H+L), Alexa Fluor 488-conjugated anti-goat IgG (H+L) and Alexa Fluor
488-conjugated anti-mouse IgM were obtained from GE Healthcare, Thermo Fisher Scientific,
Jackson ImmunoResearch, Life Technologies, Jackson ImmunoResearch, Invitrogen andInvitrogen, respectively. Anti-actin antibody (C4, mouse) and HRP-linked anti-mouse IgG wereobtained from Millipore and BioRad, respectively.

5

Results
Identification of shootin1b and preparation of its specific antibody
Immunoblot analyses of E18.5 mouse brain lysate with anti-shootin1 antibody revealed an
immunoreactive band at 87 kD, in addition to the 60-kD band corresponding to shootin1(Toriyama, et al., 2006) (Fig. 1a). Database searches identified mouse, rat and human splicevariants of shootin1 which encodes proteins of 631, 633 and 631 amino acids, respectively, witha predicted molecular mass of 71.3 kD, 71.4 kD and 71.3 kD, respectively (accession no.: mouse,
NP_001107784.1; rat, ABK56023.1; human, NP_001120683.1) (Fig. 1b and Fig. S1a). We
renamed the original shootin1 as shootin1a (accession no.: mouse, ABK56021.1; rat,
ABK56023.1; human, ABK56022.1) and refer to the novel isoform as shootin1b. In mouseshootin1b, the three amino acids ASQ at the C-terminus of shootin1a (residues 454-456) aresubstituted by 178 amino acid residues (Fig. 1c-c’ and Fig. S1b). In addition, similar proteins
have been predicted in other organisms such as Macaca mulatta (accession no.:XP_002805879.1), Canis lupus (accession no.: XP_005637827.1), Bos taurus (accession no.:XP_003587946.2), Gallus gallus (accession no.: XP_421781.3) and Xenopus (Silurana)
tropicalis (accession no.: XP_004915785.1).
We then cloned the cDNA for rat shootin1b, and raised an antibody against theshootin1b-specific region (amino acid residues 454-633) of rat shootin1b. Immunoblot analyses
with this antibody recognized rat recombinant shootin1b at 87 kD and an 87-kD band in E18.5
mouse brain lysate (Fig. 1a’), thereby demonstrating the expression of shootin1b in the embryonicbrain. This antibody did not detect the 60-kD band corresponding to shootin1a in the immunoblot
of mouse brain lysate, indicating that it recognizes specifically shootin1b. Similar data wereobtained using E18.5 rat brain lysates (data not shown).
Shootin1b is widely distributed in adult and developmental tissues
Next, we examined the expression of shootin1b in the mouse brain and peripheral tissues using
immunoblot analysis. Shootin1b was highly expressed in the embryonic brain but could not be
detected in the adult brain (Figs. 1d-d’ and S2). In contrast to the brain-specific shootin1a(Toriyama, et al., 2006), shootin1b was detected widely in adult peripheral tissues including thelung, liver, stomach, small intestine, large intestine, spleen, pancreas, kidney and skin, and inembryonic peripheral tissues including the lung, stomach, small intestine, large intestine, pancreas,kidney and skin (Figs. 1d-d’ and S2).
Localization of shootin1b in the skin
We next examined the localization of shootin1b in adult and E18.5 peripheral tissues byimmunohistochemical analyses using the shootin1b-specific antibody; shootin1b localization in6the brain will be reported separately. In adult and embryonic mouse skin, intense shootin1b
immunoreactivity was detected in all layers of the epidermis (the basal/germinal, spinous andgranular layers), which constitute a stratified squamous epithelium (Fig. 2a-a’’’ and 2b-b’’’), andin the hair follicles (Fig. 2b’’’). On the other hand, shootin1b-immunoreactive cells were sparsein the dermis (Fig. 2a-a’ and 2b-b’), and the sebaceous gland lacked shootin1b immunoreactivity(Fig. 2a’ and 2a’’’). The cornified layer showed very strong immunofluorescence; however, we
do not rule out the possibility that this involves nonspecific immunostaining, because the cornified
layer showed immunofluorescence even in the absence of the anti-shootin1b antibody (Fig. S3aand b). So, we analyzed the expression of shootin1b mRNA by RT-PCR. The shootin1b PCR
product was detected both in the cornified layer and whole skin of P5 mice (Fig. S4), suggestingthat shootin1b is expressed in the cornified layer. Interestingly, shootin1b accumulated at the cellcell contact sites of some epithelial cells in the epidermis and hair follicles (arrowheads, Fig. 2a’’’and 2b’’-b’’’).
Localization of shootin1b in the gastrointestinal tract
Figs. 3-5 show shootin1b immunoreactivity in the gastrointestinal tract. The murine stomach
consists of two functionally distinct compartments: the forestomach, which has a keratinizedsquamous epithelium, and the glandular stomach with a simple columnar epithelium (Troy, et al.,2007). In the adult and E18.5 forestomach, unkeratinized cells of the stratified squamous
epithelium were intensely stained by the anti-shootin1b antibody (Fig. 3a-a’’ and 3b-b’’).
Although the keratinized layer also showed immunofluorescence, we do not rule out thepossibility that this represents nonspecific immunostaining because of immunofluorescence in theabsence of the anti-shootin1b antibody (Fig. S3c and d). The simple columnar epithelium of theadult and E18.5 glandular stomach showed immunoreactivity for shootin1b (Fig. 4a-a’’’ and bb’’). We performed double staining of the adult glandular stomach with the antibodies against aneck cell marker secretory protein TFF2 (Syu et al., 2012) and a chief cell marker transcription
factor Mist1 (Ramsey et al., 2007). As shown in Fig. 4c-c’’’’, the neck zone which contains TFF2
positive mucous neck cells, showed relatively weak shootin1b-immunoreactivity. On the other
hand, shootin1b was expressed in Mist1-positive chief cells in the base zone (arrows, Fig. 4d’’).
The murine intestines also contain simple columnar epithelium. As shown in Fig. 5, the epitheliaof the E18.5 small intestine and the adult and E18.5 large intestine were immunoreactive forshootin1b. In some of the epithelial cells in the stomach and intestines, shootin1b accumulated at
the lateral membrane (arrowheads, Figs. 3-5).
Localization of shootin1b in other tissues
In the adult and E18.5 lung, the epithelia of the bronchus, which form a pseudostratified
7epithelium, showed strong shootin1b immunoreactivity (Fig. 6a-a’’ and b-b’’). In addition, we
observed shootin1b-immunoreactive non-epithelial cells scattered in the lung (arrows, Fig. 6b’’).
In the adult liver, strong shootin1b immunoreactivity was detected in the epithelial cells of thebile duct (Fig. 6c-c’’) which are immunoreactive for a bile duct cell marker cytokeratin-19 (Wenet al., 2011) (arrowheads, Fig. 6d’’). We also detected relatively weak immunoreactivity in cellcell contact sites of the hepatic cells (arrows, Fig. 6c’’) which are immunoreactive for a hepatic
cell marker albumin (Yadav et al., 2009) (arrowheads, Fig. 6e’’). In the adult and E18.5 pancreas(Fig. 7), the epithelial cells of the secretory duct as well as acinar cells, which are immunoreactive
for an acinar cell marker amylase (Preis et al., 2011) (arrowheads, Fig. 7c’’), were stained by theanti-shootin1b antibody. In some epithelial cells in the bile duct and the secretory duct of thepancreas, shootin1b accumulated at cell-cell contact sites (arrowheads, Figs. 6c’’ and 7a’’).
In the adult spleen, we observed shootin1b-immunoreactive non-epithelial cells (Fig. 8).
The shootin1b-immunoreactive cells also showed immunoreactivity for CD11c, a marker for
dendritic cells (Metlay, et al., 1990; Leenen, et al., 1998; McIlroy, et al., 2001) (arrows),
suggesting that the dendritic cells in the spleen express shootin1b.
Shootin1b colocalizes with E-cadherin and cortactin at cell-cell contact sites
As described above, we noted that shootin1b accumulates at cell-cell contact sites of some of theepithelial cells examined here. Recently, we reported that cortactin interacts with shootin1a at
axonal growth cones (Kubo, et al., 2015); shootin1b also contains the region responsible for theinteraction with cortactin (amino acid residues 261-377 of shootin1a). In addition, cortactin
accumulates at cadherin-based cell-cell contacts and is thought to regulate cell adhesions (ElSayegh, et al., 2004; Helwani, et al., 2004; Ren, et al., 2009; Truffi, et al., 2014). Finally, therefore,
we analyzed the colocalization of shootin1b with E-cadherin and cortactin in epithelial cells intissues and cultured cells. As shown in Fig. 9, shootin1b colocalized with E-cadherin and cortactin
at cell-cell contact sites of epithelial cells of the epidermis and glandular stomach (arrowheads).
In addition, shootin1b colocalized with E-cadherin and cortactin at cell-cell contact sites ofcultured EpH4 epithelial cells, which are derived from mouse mammary glands (Fig. 10,
arrowheads).

8

Discussion
Previous studies reported that shootin1a (formerly shootin1) mediates key processes in neuronalcells, including neuronal symmetry-breaking (Toriyama, et al., 2010; Inagaki, et al., 2011),
polarity formation (Toriyama, et al., 2006; Sapir, et al., 2013), signal-mediated axon outgrowth(Toriyama, et al., 2013; Kubo, et al., 2015) and axonal transport (Katsuno, et al., 2015).
Mechanically, shootin1a acts as a clutch molecule to couple actin filaments and cell adhesions,
thereby generating the force underlying these processes (Shimada, et al., 2008; Katsuno, et al.,2015; Kubo, et al., 2015). In this study, we identified shootin1b, a novel splicing isoform ofshootin1a. Shootin1b contains almost the entire amino acid sequence of shootin1a, including thethree coiled-coil domains and a proline-rich region, and an additional C-terminal 178-residue
region that is specific to shootin1b (Fig. 1c’). In contrast to shootin1a, which is detected only inthe brain (Toriyama, et al., 2006), shootin1b was distributed not only in the brain but also in thelung, liver, stomach, small intestine, large intestine, spleen, pancreas, kidney and skin (Fig. 1dd’). These data expand our view of shootin1-mediated cellular processes.
The present study showed that shootin1b accumulates at cell-cell contact sites of someepithelial cells, where it colocalizes with E-cadherin and the actin-binding protein cortactin.
Recently, we found that cortactin interacts with shootin1a, as another clutch molecule, andmediates shootin1a-dependent axon outgrowth. Shootin1b also contains the region responsible
for the interaction with cortactin (amino acid residues 261-377 of shootin1a) (Kubo, et al., 2015).
Cortactin accumulates at cadherin-based cell-cell contacts and is thought to regulate celladhesions (El Sayegh, et al., 2004; Helwani, et al., 2004; Ren, Helwani, et al., 2009; Truffi,
Dubreuil, et al., 2014). The possible involvement of shootin1b in cell adhesions remains animportant issue for future studies.
Shootin1b was also detected in non-epithelial cells including dendritic cells.

Asclutch molecules are thought to produce forces not only for axon outgrowth but also for cell
migration (Suter and Forscher, 2000; Thievessen, et al., 2013; Craig, et al., 2015; Kubo, et al.,2015), shootin1b may play a role in the migration of non-neuronal cells. The mRNAs encoding
shootin1a and shootin1b are derived from the same premature RNA (Fig. S1a). The mechanism
regulating the differential production of mRNAs for shootin1b and shootin1a in different cell
types is an intriguing problem that would merit further investigations.

Acknowledgements
EpH4 cells were the kind gift of Dr. E. Reichman. We thank Drs. Sadao Shiosaka and Michinori
Toriyama for reviewing the manuscript.

9Funding
This research was supported in part by JSPS Grant-in-Aid for Scientific Research on Innovative
Areas (25102010), JSPS KAKENHI (26290007), Osaka Medical Research Foundation for
Incurable Diseases and Mitsubishi Foundation.

10

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13

Figure Legends
Fig. 1 Identification and tissue distribution of shootin1b. (a-a’) Immunoblot analyses of E18.5
mouse brain lysate (10 μg proteins), purified recombinant shootin1a (5 ng) and purified
recombinant shootin1b (5 ng) with anti-shootin1 antibody (a) and anti-shootin1b antibody (a’).
Anti-shootin1 antibody reacts with shootin1a and shootin1b, while anti-shootin1b antibody
recognizes specifically shootin1b. (b) Amino acid sequences of mouse, rat, and human shootin1b.
Residues that are identical in all three sequences are boxed. (c-c’) Schematic representations ofmouse shootin1a (c) and shootin1b (c’), showing three coiled-coil domains (CC1–3) and a single
proline-rich region. Amino acid residues 454-631 are the shootin1b-specific region. (d-d’)
Immunoblot analyses of shootin1b in adult mouse tissues (10 g proteins) (d) and E18.5 mouse
tissues (10 g proteins) (d’) with anti-shootin1b antibody. Immunoblots with anti-actin antibody
served as loading controls.
Fig. 2 Immunohistochemical localization of shootin1b in the mouse skin. Shootin1b
immunoreactivity (green) and DAPI staining (red) in adult (a-a’’’) and E18.5 (b-b’’’) mouse skin.
a’’, a’’’, b’’ and b’’’ are enlarged views of the areas 1, 2, 3 and 4, respectively. Shootin1b is locatedin all layers of the epidermis (Epi) and hair follicles (HF). BL, basal layer; CL, cornified layer;
GL, granular layer; SG, sebaceous gland; SL, spinous layer. Arrowheads indicate shootin1b
accumulation at cell-cell contact sites. Bars: (a’ and b’) 30 μm; (a’’, a’’’, b’’ and b’’’) 10 μm.
Fig. 3 Immunohistochemical localization of shootin1b in the mouse forestomach. Shootin1b
immunoreactivity (green) and DAPI staining (red) in the adult (a-a’’) and E18.5 (b-b’’) mouseforestomach. a’’ and b’’ are enlarged views of the areas 1 and 2, respectively. KL, keratinizedlayer. Shootin1b is localized in the unkeratinized squamous epithelium. Arrowheads indicate
shootin1b accumulation at cell-cell contact sites. Bars: (a’ and b’) 30 μm; (a’’ and b’’) 10 μm.
Fig. 4 Immunohistochemical localization of shootin1b in the mouse glandular stomach. (aa’’’ and b-b’’) Shootin1b immunoreactivity (green) and DAPI staining (red) in the adult (a-a’’’)and E18.5 (b-b’’) glandular stomach. Base, base zone; Neck, neck zone. (c-c’’’’ and d-d’’)Immunohistochemical colocalization of shootin1b (green) with a neck cell marker secretory
protein TFF2 (red) (c-c’’’’) and a chief cell marker transcription factor Mist1 (red) (d-d’’) in theadult glandular stomach. Blue indicates DAPI staining. a’’, a’’’, b’’ and c’-c’’’’ are enlarged viewsof the areas 1, 2, 3 and 4, respectively. Arrows in d” indicate shootin1b expression in Mist1positive mucous neck cells, while arrowheads in a’’’ and b’’ show shootin1b accumulation at cellcell contact sites. Bars: (a’, b’ and c) 30 μm; (a’’, a’’’, b’’ and d’’) 10 μm; (c’’’’) 20 μm.

14Fig. 5 Immunohistochemical localization of shootin1b in the mouse intestines. Shootin1b
immunoreactivity (green) and DAPI staining (red) in the E18.5 mouse small intestine (a-a’’),adult large intestine (b-b’’) and E18.5 large intestine (c-c’’). a’’, b’’ and c’’ are enlarged views of
the areas 1, 2 and 3, respectively. Arrowheads in a’’ indicate shootin1b accumulation at the lateral
membrane. Bars: (a’, b’ and c’) 30 μm; (a’’, b’’ and c’’) 10 μm.
Fig. 6 Immunohistochemical localization of shootin1b in the mouse lung and liver. (a-a’’, bb’’ and c-c’’) Shootin1b immunoreactivity (green) and DAPI staining (red) in the mouse adultlung (a-a’’), E18.5 lung (b-b’’) and adult liver (c-c’’). a’’, b’’, and c’’ are enlarged views of theareas 1, 2 and 3, respectively. BD, bile duct; HC, hepatic cells; PV, portal vein; RE, respiratory
epithelium. (d-d’’ and e-e’’) Immunohistochemical colocalization of shootin1b (green) with a bile
duct marker cytokeratin-19 (red) (d-d’’) and a hepatic cell marker albumin (red) (e-e’’) in theadult liver. Arrows in (b’’) and (c’’) indicate non-epithelial cells in the lung and cell-cell contact
sites of hepatic cells, respectively. Arrowheads in (c’’) indicate cell-cell contact sites of bile ductcells. Arrowheads in (d’’) and (e’’) indicate shootin1b expression in cytokeratin-19-positive bile
duct cells and albumin-positive hepatic cells, respectively. Bars: (a’, b’ and c’) 30 μm; (a’’, b’’,c’’ and e’’) 10 μm; (d’’) 5μm.
Fig. 7 Immunohistochemical localization of shootin1b in the mouse pancreas. (a-a’’ and bb’’) Shootin1b immunoreactivity (green) and DAPI staining (red) in the mouse adult pancreas (aa’’) and E18.5 pancreas (b-b’’). a’’ and b’’ are enlarged views of the areas 1 and 2 respectively.
PC, parenchymal cells of the pancreas; SD, secretory duct of the pancreas. (c-c’’)Immunohistochemical colocalization of shootin1b (green) with an acinar cell marker amylase(red) in the adult pancreas. Arrowheads in (c’’) indicate shootin1b expression in amylase-positive
acinar cells. Bars: (a’and b’) 30 μm; (a’’, b’’ and c’’) 10 μm.
Fig. 8 Immunohistochemical localization of shootin1b in the mouse spleen. Shootin1b
immunoreactivity (green), CD11c-immunoreactivity (red) and DAPI staining (blue) in the adultmouse spleen. b-b’’ and c-c’’ are enlarged views of the areas 1 and 2, respectively Arrows indicate
co-localization of shootin1b with the dendritic cell marker CD11c. Bars: (a’’) 30 μm; (b’’ andc’’) 10 μm.
Fig. 9 Shootin1b colocalizes with E-cadherin and cortactin in epithelial cells of the skin andglandular stomach. (a-a’’’’’ and b-b’’’’’) Immunohistochemical colocalization of shootin1b(green) with E-cadherin (red) (a-a’’’’’) and cortactin (red) (b-b’’’’’) in E18.5 mouse skin. (c-c’’’’’and d-d’’’’’) Immunohistochemical colocalization of shootin1b (green) with E-cadherin (red) (c15c’’’’) and cortactin (red) (d-d’’’’) in the E18.5 mouse glandular stomach. a’’’-a’’’’’, b’’’-b’’’’’, c’’’c’’’’’ and d’’’-d’’’’’ are enlarged views of the indicated areas. Arrowheads indicate colocalizationof shootin1b with E-cadherin and cortactin at cell-cell contact sites. Bars: (a’’, b’’, c’’ and d’’) 30
μm; (a’’’’’, b’’’’’, c’’’’’ and d’’’’’) 5 μm.
Fig. 10 Shootin1b colocalizes with E-cadherin and cortactin at cell-cell contact sites ofcultured EpH4 cells. Immunocytochemical colocalization of shootin1b (green) with E-cadherin(a-a’’) and cortactin (b-b’’) in cultured EpH4 cells. The lower panels show vertical sections of
the region indicated by the dotted lines. Arrowheads indicate colocalization of shootin1b with Ecadherin and cortactin at cell-cell contact sites. Bars: 20 m.
Electronic Supplementary Material
Fig. S1 Two splice variants and isoforms of the mouse shootin1 gene (a). The structure of theshootin1 locus and transcripts. (i) The structure of the mouse shootin1 genomic locus. (ii)Transcripts in the shootin1 locus. The shootin1 gene has two splice variants, a 3767-nt mRNA
(NM_175172.4) encoding a protein of 456 amino acids and a 4081-nt mRNA (NM_001114312.1)
encoding a protein of 631 amino acids. Gray and white boxes indicate exons and untranslated
regions, respectively. Positions of stop codons (TAA and TGA) are shown. (b) Alignment ofshootin1 isoforms. The amino acid sequences were obtained from the following sources:
shootin1a (ABK56021.1) and shootin1b (NP_001107784.1).
Fig. S2 Immunoblot analyses of shootin1b in adult mouse tissues and E18.5 mouse tissues
with anti-shootin1b antibody.
Whole scans of the immunoblotted membranes of Fig. 1d are shown.
Fig. S3 Control staining of the skin and forestomach without Anti-shootin1b Antibody. Two
serial sections of the adult (a) and E18.5 (b and e) mouse skin, and the adult (c) and E18.5 (d)forestomach were immunostained: one section stained with anti-shootin1b antibody (shootin1b)
and the other stained without the antibody (negative control). They were also co-stained withDAPI (a-d), anti-E-cadherin antibody (e) and anti-cortactin antibody (e) (red). CL, cornified
layer; KL, keratinized layer. The data in (e) represent control data of those in Fig. 9. Bars: 50 m.
Fig. S4. RT-PCR analysis of shootin1b mRNA in the skin.
RNA was extracted from the cornified layer and whole skin of P5 mouse. PCR was carried out
by using shootin1b-specifc primers (left) and -actin-specific primers (right) as a positive control.
The PCR products were electrophoresed on a 2% agarose gel. The expected 233-bp shootin1b
16

and 165-bp -actin PCR products were detected both in the cornified layer (lane 1) and whole
skin (lane 2). M, 1 kb DNA ladder marker.

Further Reading
Ergin V, Erdogan M, Menevse A (2015) Regulation of shootin1 gene expression involves NGFinduced alternative splicing during neuronal differentiation of PC12 cells. Sci Rep 5:17931
After the submission of this paper, a report by Ergin et al. (2015) was published. This paper
describes shootin1b expression in a PC12 cell line.

17Fig. 1ab

IB: anti-shootin1

(KD)
220
160
120
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Shootin1b
Shootin1a40

a’ IB: anti-shootin1b

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Shootin1b

40

c

1 7

Shootin1a NH2

c’

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CC1
1 7

Shootin1b NH2

59

59

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137

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233 259CC2

353 369

COOH

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454

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Actin

Adult

d’

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Fig. 2

a

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Epi

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GL

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a

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b
E18.5

KL

b’
2

KL

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Fig. 4

Lumen

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a’

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Base Neck

Adult

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b’

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b’’

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d

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a
1

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a’
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1

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Shootin1bd’ Cytokeratin-19 d’’

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Albumine’’Fig. 7

1

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Pancreas

PC

b

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c
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Adult

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Shootin1bc’SDAmylase

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Fig. 8aShootin1b

a’

CD11c

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Shootin1b/ CD11c / DAPI
1

b

b’

b’’

c

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Fig. 9a’a’’

Shootin1b

E-cadherin

Merge

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Merge

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Cortactin

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a’’’

a’’’’

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E18.5
E18.5

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a

Fig. 10aShootin1ba’

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Merge

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Figure S1a(i)shootin1
Exon 1

3

4 5

6 7 8

9 10 11 12 13 14 15 16

17

(ii)

ATG

TAA

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558

1928

1

b

2

ATG

TGA

558

2453

3767

4081

Figure S2
Adult
IB:
anti-shootin1b

Shootin1b

E18.5

Figure S3

Shootin1b / DAPI

CL

b

CL

Shootin1b / DAPI

KL

d

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e

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Forestomach

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DAPI

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Shootin1b

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MergeFigure S4

M

300 bp200 bp1

M

2

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200 bp100 bp12

‐actin