Supplementary Figures
1
2
Supplementary Figure Legends
Figure S1. Cre expression pattern in the uterine epithelia. (A-D) K5Cre expression in the
uterine epithelia from E16.5 to postnatal days. (A) At E16.5, K5Cre expression is observed
in a few numbers of epithelial cells. (B) K5Cre expression in the uterine epithelium is
broader in the uterine epithelium as compared to E16.5. (C-D) At postnatal stages, K5Cre is
expressed in most cells of the uterine epithelia. Magnification: (A-D) x100. Bar: 200 m.
Figure S2. Formation of stratified uterine epithelia in the LOF mutants of-catenin
grafted in non-ovariectomized hosts. (A, B) Hematoxylin and eosin (H&E) staining of
control and K5Cre;Catnbfl/fl mutant uteri grafted for 4 weeks in non-ovariectomized nude
mice. (A-A’) Control uteri exhibit single-layered, columnar epithelium and UGs. (B-B’) In
K5Cre;Catnbfl/fl mutant uteri, the epithelium becomes stratified layer with squamous cell
shape. (C-C’, D-D’) Immunostaining analysis indicates loss of catenin expression in the
mutant uterine epithelium. (E-E’, F-F’) Cyclin D1 expression is reduced in the mutant uterine
epithelium (green arrowheads). (G-G’, H-H’) Foxa2 expression is also reduced in the mutant
uterine epithelium. Mutant uterine epithelium is altered into a stratified epithelium as shown
by the expressions of (I-I’, J-J’) Keratin 14 and (K-K’, L-L’) p63, which are markers for
stratified epithelia. Magnification: (A-L) x40; (A’-L’) x400, higher magnification of the
boxed areas. Bars: 500 m and 50 m. UG: uterine glands.
Figure S3. Average glandular number and cell proliferation rate in the GOF mutants
of-catenin grafted in non-ovariectomized hosts. (A) Average number of glands in the
GOF mutants of-catenin (n=3). The number of glands in the mutant uterine grafts is
significantly increased as compared with the control uterine grafts. (B-C) The cell
proliferation rate (%) in the mutant uterine grafts is significantly increased as shown in the
graphs of (B) pH3 expressing cells and (C) Ki67 expressing cells. Data are presented as
3
mean ± s.e.m. of three replicates in three independent experiments. Statistical significance
was calculated using the Student’s t-test followed by F-test (*p < 0.05).
Figure S4. Normal mRNA expression levels in different endometrial cell lines. mRNA
expression levels of -catenin, Foxa2, cyclin D1 in UE2AA, Ishikawa, ECC-1 and ARK-1
cell lines. -actin is utilized as endogenous control for human cell lines such as Ishikawa,
ECC-1 and ARK-1 while mL8 is utilized for the mouse endometrial cell line UE2AA.
Figure S5. Knockdown of Foxa2 in different endometrial cell lines.
Western blot
analyses show the reduced expression of Foxa2 after transfection with Foxa2 siRNA in
UE2AA, Ishikawa, ECC-1 and ARK-1 cell lines. Western blot analyses show the augmented
expression of Foxa2 after transfection with Foxa2 overexpression vector in UE2AA,
Ishikawa and ECC-1 cell lines. OE: overexpression. Red arrow indicates the molecular
weight of the Foxa2 protein. -tubulin is utilized as endogenous control.
Figure S6. Cell cycle analyses in UE2AA, ECC-1 and ARK-1 cell lines after the
knockdown of Foxa2.
(A) UE2AA cell line shows increased percentages of cells
undergoing G1 phase and decreased percentages of cells undergoing S and G2/M phases of
the cell cycle. (B) Cell cycle analysis in ECC-1 shows no differences in the percentages of
cells undergoing G1, S and G2/M phases of the cell cycle. (C) ARK-1 cell line shows
increased percentages of cells undergoing G1 phase and decreased percentage of cells
undergoing S and G2/M phases of the cell cycle.
4
Supplementary Materials and Methods
Human specimens
Tissue specimens were obtained from three eutopic endometria specimens, 28 complex
atypical endometrial hyperplasia specimens, 23 G1 and 18 G3 EACs specimens. mRNA
samples were obtained from six eutopic endometria and six complex atypical endometrial
hyperplasia human specimens. The human specimens were frozen at the time of surgery at
the Keio University Hospital after obtaining patients' informed consent. All tumors were
histologically classified and further analyzed by experienced gynecological pathologists in
Keio University Hospital using standard World Health Organization criteria (Hirasawa et al
2003).
Mice and Uterine Grafting
The Catnbfl/fl (Huelsken et al 2001), Catnb(ex3)fl/+ (Harada et al 1999), Keratin (K)
5Cre (Tarutani et al 1997), and ROSA26R (Soriano 1999) strains were used in this study.
Adult female nude mice were purchased from Charles River (Wilmington, MA). Renal
capsule grafting was conducted as described previously (Cunha 1976, Kurita et al 2004). For
embryonic sampling, pregnant females were sacrificed on embryonic day (E) 18.5 and the
uteri were isolated and dissected into halves and prepared for renal capsule grafting. Fourweek-old female nude mice were ovariectomized and maintained for two weeks to reduce
systemic ovarian steroid hormones to basal levels. Renal capsule grafting of the uteri was
performed two weeks after ovariectomy or in non-ovariectomized nude mice. After two
weeks of rest, the nude mice were weighed and anesthetized with Pentobarbital Sodium Salt
(Tokyo Chemical Industry Co. LTD, Japan) solution. The back skin at the area of incision
was swabbed with 70% ethanol. Incision along the midline was made at the back skin and the
mouse was placed on its side while locating the kidney through the body wall. Small incision
5
was then made in the body wall just enough for the size of the kidney. The kidney was
popped out of the hole and gently made an incision (or pocket) between the capsule and the
parenchyma of the kidney. The uterine grafts were inserted in these pockets. In host nude
mice, the left kidney was designated for control grafts while the right kidney was designated
for mutant graft to insure the same hormonal and growth conditions for the control and
mutant uteri transplants. After grafting, the kidney was eased back into the body cavity. The
incision of the body cavity was closed and sutured and the back skin was also closed with the
aid of wound clip. The animals were sacrificed 4 weeks after renal capsule grafting. (Detailed
procedure and visual presentation of renal capsule grafting is available in this site
http://mammary.nih.gov/tools/mousework/Cunha001/index.html).
All animal experiments were performed according to the regulations set by the animal
study committee of Wakayama Medical University, Wakayama, Japan and Kumamoto
University, Kumamoto Japan.
Cell lines
The human uterine adenocarcinoma cell line, ECC-1, was obtained from American
Type Culture Collection (ATCC). ECC-1 cell line was maintained in RPMI-1640 medium
(Wako, Osaka, Japan) with 5% Fetal Bovine Serum (FBS). ECC-1 has been authenticated by
the ATCC. Ishikawa cell line another human-endometrial adenocarcinoma cell line of
epithelial origin was obtained from European Collection of Cell Cultures (ECACC).
Ishikawa cell line was maintained in Dulbecco’ Modified Eagle Medium, DMEM (Wako,
Osaka, Japan) with 10% FBS. Ishikawa cell line was authenticated by ECACC and revalidated its DNA profile with Short Tandem Repeat analysis by Japanese Collection of
Research Bioresources, Tokyo, Japan. Uterine papillary serous adenocarcinoma (UPSC)
ARK-1 cell line was obtained from Dr. Allesandro Santin (Guzzo et al 2012). ARK1 was
maintained in RPMI-1640 medium (Wako, Osaka, Japan) with 10% FBS, 1% Penicillin
6
Streptomycin Solution and 0.3% fungizone, antimycotic solution (Invitrogen catalog No.
15290-018). Mouse endometrial cell line, UE2AA, was obtained from Dr. Yasuhiro
Tomooka (Hanazono et al 1997). UE2AA cell line was maintained in DMEM/F12 (GIBCO,
Tokyo, Japan) with 10% FBS and supplemented with 1% Insulin-Transferrin Selenium (ITS)
solution. Cells were maintained at 37oC in an atmosphere containing 5% CO2.
Histology, LacZ staining and Immunohistochemistry
Hematoxylin and eosin staining (H&E) and immunohistochemistry were performed
by standard procedures as previously described (Haraguchi et al 2007). For H&E staining,
the samples were embedded in the paraffin and cut using the microtome with 6m thickness.
To analyze the K5Cre expression in the uterine epithelia, we utilized K5Cre;Rosa26R system
(Ahn and Joyner 2004). The K5Cre male mice were crossed with female Rosa26R Creindicator (R26R) mice to obtain K5Cre;R26R/R26R female offspring mice. LacZ staining
was performed as previously described (Haraguchi et al 2007, Phippard et al 1999). Tissue
sections were stained with primary antibodies as shown in the table below. Final
visualization was done by means of the streptavidin-biotin system using diaminobenzine as
final chromogen. Tissue sections were counterstained with Hematoxylin staining for 10
seconds.
Immunostaining of -CATENIN, LEF-1 and FOXA2 in human specimen were
scored by three different Researchers who were blinded to the study. -CATENIN, LEF-1
and FOXA2 slides were scored for the presence and absence of nuclear staining pattern. catenin scoring for each slide was determined according to the previous study (Scholten et al
2003). Histological score was obtained from each sample which ranged from 0 (no staining)
to 3 (intense staining) and subjected to non-parametric analysis utilizing the Wilcoxon ranksum test.
7
Immunofluorescence was visualized using Alexa Fluor 488 or 546 IgG against
primary antibodies (Molecular Probes/Invitrogen, Carlsbad, CA). Nuclear counterstaining for
immunofluorescence was performed with Hoechst 33342 (Sigma-Aldrich, Munich,
Germany). The cell proliferation rate (%) was determined by selecting three fields, counting
the number of proliferating cells, and the subsequent calculations were calibrated with the
total number of epithelial cells (1 000 cells) within three objective fields (200x). Experiments
to determine the proliferation was repeated at least three replicates in three independent
experiments. Photomicrographs were taken using the Olympus BX50 microscope and
Olympus FV1000D confocal microscope (Olympus, Tokyo, Japan).
Plasmid construction and Transfection of cell lines
The -1,800/+79 bp DNA fragments of Foxa2 were cloned by PCR using mouse
genomic DNA and were inserted into the pGL3 basic vector (Promega, Madison, WI). Full
length of mouse Foxa2 was kindly provided by Dr. H. Sasaki (Kumamoto University, Japan).
Generation of overexpression of mouse FoxA2 was done by ligating the coding sequence of
mouse FoxA2 gene into pCS2+MT vector (Rupp et al 1994, Turner and Weintraub 1994). catenin overexpression vector was kindly provided by Drs. T. Yamaguchi, Polakis, O. Tetsu
and F. McCormick. Cells were split 2 days before transient transfection and grown to 80%
confluency. Transfection of cell line was performed using the Lipofectamine 2000
(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Cells were harvested
after 48 hours and were screened for gene expression. For Luciferase assay, the firefly
luciferase reporter constructs were transfected at 0.5 ug/plate along with 0.1 ug/plate of the
Renilla (pRL-tk) reporter to permit normalization of transfection efficiencies. After 24 hours,
cells were harvested in 1x passive lysis buffer (Promega, Madison, WI). Luciferase activity
was measured by a Monolight 2010 luminometer utilizing the Dual-Luciferase Reporter
Assay (Promega, Madison, WI) as indicated by the manufacturer. Luciferase Assay data
8
were presented as mean ± s.e.m. of more than three independent experiments to ensure
reproducibility. Statistical analyses were performed using One-Way ANOVA followed by
Bonferroni’s multiple comparison tests with the alpha level at 0.05. Primers used for PCR
cloning
of
Foxa2
promoter
region:
AAACTCGAGGCGTAGTAGCTGCTCCA
-
Foxa2
3';
Foxa2
Forward:
5'
Reverse:
5'
–
–
TCCAAGCTTAAAAACGCCGGCAAGAAC - 3'. Primers used for mutagenesis of the
TCF/LEF binding sites: Site 1 mutation: 5' – CGTAACTAACGCAGCCAGGGCAGT- 3';
Site 2 mutation: 5’ – CGGTGTGTCCAGGGTACTTTTCAGT- 3’; Site 3 mutation: 5’ –
TTGGTGGCCTGTAGCGTGAGGCAA-
3’
and
Mismatch
mutation:
5'
–
TGGGCCTTGTGGGATGATCACCGT- 3'. Red color indicates the sites of mutation.
Semi-quantitative and Quantitative Real- time PCR
Endometrial cell lines were cultured in six-well-cultured plates (5 x 105 cells/well)
and maintained in its designated media. Total RNA was extracted from the cell lines after 48
hours of culture or transfection. All RNA samples were reverse transcribed using the
Superscript II first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) with
oligomer random primers (Takara, Japan). One microliter of the synthesized cDNA were
mixed with AmpliTaq Gold polymerase (Applied Biosystems, NJ) for PCR. The expression
level of each amplicon was calculated by normalizing each cDNA to -actin or mL8 as
specified in the text. Two microliters of the synthesized cDNA were mixed with SYBR
Green PCR master mix (Applied Biosystems, CA) for real time PCR. The expression level of
each amplicon was calculated by normalizing each cDNA to E-cadherin and then calculated
as the fold change compared with the control.
Primers for Human sequences used were the following: -catenin Forward: 5'GCCGGCTATTGTAGAAGCTG
-
3';
9
-catenin
Reverse:
5'-
GAGTCCCAAGGAGACCTTCC
-
3'
(Park
et
al
2008).
Foxa2
Forward:
5'-
ACACCACTACGCCTTCAACC - 3'; Foxa2 Reverse: 5'- GTGCATCACCTGTTCGTAGG 3' (Tang et al 2011). Cyclin D1 Forward: 5'- GAGCTGCTCCTGAACAAG - 3'; cyclin D1
Reverse: 5'- TTCAATGAAATCGTGCGGG - 3' (Suriano et al 2005). E-cadherin Forward:
5'-
GACCAGGACTATGACTACTTGAAACG
-
3';
E-cadherin
Reverse:
5'-
ATCTGCAAGGTGCTGGGTGAACCTT- 3' (Liu et al 2005). -actin Forward: 5'GATGAGATTGGCATGGCTTT- 3'; -actin Reverse: 5'- CACCTTCACCGTTCCAGTTT3' (Santra et al 2006).
Primers for mouse sequences used were the following: -catenin Forward: 5'GCCTGTAGAGTTGCTGAGAGGGCT-
3';
-catenin
Reverse:
5'-
TCACTGCGTGAGTCACTCCCCAAA - 3' (Chen et al 2012). Foxa2 Forward: 5'CCCTGGCTGCAGACACTTCCTACT-
3';
Foxa2
Reverse:
5'-
GAGGCCTGAAGTGTGGTGGCTACT- 3' (Li et al 2012). Cyclin D1 Forward: 5'GATGGCGATCGTCCTGTCATGCTG
-
3';
cyclin
D1
Reverse:
5'-
ACCGGAGACTCAGAGCAAATCCCC- 3' (Lammie et al 1992). mL8 Forward: 5'ACAGAGCCGTTGTTGGTGTTG-
3';
mL8
Reverse:
5'-
CAGCAGTTCCTCTTTGCCTTGT- 3 (Miyagawa et al 2009).
Transfection of siRNA
Control (ON-TARGET plus Non-targeting siRNA) and Foxa2 double stranded
siRNAs (ON-TARGET plus Smart pool siRNA) were chemically synthesized (Dharmacon,
Lafayette, CO, USA). Concentrations of Foxa2 siRNA were evaluated using the Negative
Control siRNA Alexa Fluor 546 (Qiagen, Cambridge, MA) in endometrial cell lines.
Ishikawa and ECC-1 cell lines were transfected with 25 nM of Foxa2 siRNA. UE2AA was
transfected with 40 nM of Foxa2 siRNA. ARK-1 cell line was transfected with 30 nM of
10
Foxa2 siRNA. The cells were plated on six-well plates and cultured in its corresponding
media. After 24 hours, the cells were transfected with Foxa2 siRNA using the Lipofectamine
RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Knockdown of Foxa2 was evaluated using the quantitative real-time PCR (qPCR),
immunostaining and western blotting. ON-TARGET plus Smart pool Human Foxa2 siRNA
sequences were the following:
Foxa2 siRNA 1: GAACAUGUCGUCGUACGUG;
Foxa2 siRNA 2: GCAGAUACCUCCUACUACC;
Foxa2 siRNA 3: AAAUGGACCUCAAGGCCUA;
Foxa2 siRNA 4: GAACACCACUACGCCUUCA.
ON-TARGET plus Smart pool Mouse Foxa2 siRNA sequences were the following:
Foxa2 siRNA 1:CCAAACCUCCCUACUCGUA;
Foxa2 siRNA 2:UGAAUGGCAUGAACACAUA;
Foxa2 siRNA 3: GGAAUGAGCCCGUCGCUAG;
Foxa2 siRNA 4: CAAGCGAGGUGGCCUAAGC.
Chromatin immunoprecipitation (ChIP) and Pull-down assays
For ChiP assay, 12 pairs of uterine samples were dissected from Postnatal day 9 (P9)
female embryos. ChIP Assay Kit (Millipore, NY, USA) was used in this study. -catenin
(Santa Cruz, CA, USA), Foxa2 (Santa Cruz, CA) and acetyl-histone 3 (Upstate, Lake Placid,
NY, USA) antibodies (2 g each) were used. Rabbit immunoglobulin (Dako, Denmark)
antibody was used for mock control. For pull-down assay, 20 uterine samples from P9
female embryos were used. Wild Type (WT) or mutated Sites fragment bound to
Streptavidin resin (Strategen, Santa Clara, CA) was incubated with 20 uterine samples lysed
in the buffer (50 mM Tris·HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM
11
PMSF, protease inhibitor mixture). The beads were washed and analyzed with Western
blotting against anti--catenin antibody. Experiments were performed in three independent
experiments. For -catenin chip assay, the primers used were the following: Region 1:
Forward
1:
5’-
–
CACCTCAGTCGGCCTGGA
GAAGTCATCCCACAAGGCCCA
GAGTCCCTTCCTTTACGTCCAC
–
3’.
–
3’;
Reverse
1:
5’-
Forward
2:
5’-
Region
2:
3’;
Reverse
2:
5’-
GAGGAAACCCGAGATAAATAAATC – 3’. For cyclin D1 chip assay, the primers used
were the following: Forward: 5’- GGGGCGATTTGCATTTCTAT- 3’; Reverse: 5’CGGTCGTTGAGGAGGTTGG- 3’(Yang et al 2011).
Primers for pull-down assay were the following: Site 1 WT Forward: 5’CGTAACTAAAACAAACAGGGCAGTAGGTGGCCACCTACTGCCCTGTTTGTTTTAGTTACG-
3’;
3’;
Site
1
WT
Reverse:
5’-
Site 1 Mutated Forward:
5’-
CGTAACTAGCATAGGCAGTTCAGTAGGTGG- 3’; Site 1 Mutated Reverse: 5’CCACCTACTGAACTGCCTATGCTAGTTACGGGCCTCGGTGTTTCAAGGTTACTTTTCAG-
3’;
3’;
Motif
Motif
26
26
WT
Forward:
5’-
WT
Reverse:
5’-
CTGAAAAGTAACCTTGAAACACCGAGGCC- 3’; Motif 26 Mutated Forward: 5’GGCCTCGGTGTTGTACGTTTACTTTTCAG- 3’; Motif 26 Mutated Reverse: 5’CTGAAAAGTAAACGTACAACACCGAGGCC-
3’;
Motif
27
WT
Forward:
5’-
CTTGGTGGCCTGCAAAGCGAGGCAAGCCAG- 3’; Motif 27 WT Reverse: 5’CTGGCTTGCCTCGCTTTGCAGGCCACCAAG- 3’; Motif 27 Mutated Forward: 5’CTTGGTGGCCTTTAGATCGAGGCAAGCCAG- 3’ and Motif 27 Mutated Reverse: 5’CTGGCTTGCCTCGATCTAAAGGCCACCAAG- 3’. Red color indicates the sites of
mutation.
12
Western Blotting
Western blotting was performed according to previous works (Terabayashi et al
2007). Protein bands were visualized using ChemiDoc™ XRS+ System with Image Lab™
Software (BioRad, CA). Precision Plus™ Protein marker was used to identify the specific
bands of the protein of interest (BioRad, CA). Experiments were performed in three
independent experiments.
Flow cytometric analysis of cell cycle kinetics
Cells were transfected with FoxA2 siRNA in culture plates and grown for 48 hours.
Cells were harvested and stained with Propidium Iodide (PI) solution (10% Sodium citrate,
10% Triton-100, 1mg/ml PI, 10mg/ml RNAse A) for one hour. 106 cells were filtered and
examined by FACS Canto and DNA histograms were analyzed by FlowJo software (Becton
Dickinson, Franklin Lakes, NJ). Experiments were performed in three independent
experiments and the results are presented as average percentage of cells in each phase of the
cell cycle.
Whole Genome RNA Sequencing
Ishikawa cell line was cultured in six-well-cultured plates (5 x 105 cells/well) and
maintained in its designated medium. Total RNA was extracted from the cell line after 48
hours of culture or transfection. Genome Analyzer GAIIx (Illumina, San Diego, CA) was
used for whole genomic RNA sequencing and performed as described previously (Langmead
et al). RNA libraries were generated by the modified Illumina protocol using the mRNA
sequence preparation kit. Briefly, 1 g of total RNA was enriched for polyA RNA by two
successive rounds of oligo(dT) selection. The polyA RNA was then fragmented and first
strand cDNA synthesis was performed using random hexamer priming. Following second
strand cDNA synthesis, dsDNA was treated using T4 DNA polymerase, Klenow enzyme,
13
and T4 polynucleotide kinase (PNK) (New England Biolabs, Massachussetts, USA),
followed by treatment with Klenow exo- to add an A base to the 3’ end. After ligation of the
Solexa adaptor using Takara Ligation Mix (Takara, Japan), the adaptor-ligated DNAs were
amplified using Solexa PCR primers for 18 cycles, and the amplified library was isolated
from an agarose gel. The samples were purified using the QIAquick MinElute Kit (Qiagen,
Valencia, CA, USA) at each preparation step.
Whole Genome RNA Sequencing Data Analysis
For each sample, cDNA was sequenced (single 36 bp read) using the Illumina
Genome Analyzer GAIIx. The base-called sequences were obtained using SCS2.7 from RNA
sequence image data. To calculate the total amount of the transcripts of each mRNA, a series
of programs – Bowtie (Langmead et al 2009), TopHat (Trapnell et al 2010), and Cufflinks
(Trapnell et al 2010)- were used. Briefly, RNA sequence reads were mapped against the
whole reference genome (hg19) using Bowtie. The reads that did not align to the genome but
were mapped to potential splice junctions by TopHat were considered to bridge splice
junctions. The quantification of transcripts with normalized gene lengths were performed
with Cufflinks. All of the parameters except ‘Max multihits’ (TopHat) were substituted with
default options.
Statistical Analysis
Student’s t test was used to compare the means of two groups followed by F-test.
Analyses of Variance (ANOVA) was used to compare the means of three or more groups
followed by Bonferroni’s test to correct for multiple comparisons. Immunohistochemistry
staining of complex atypical endometrial hyperplasia, G1 and G3 human samples were
compared using the Wilcoxon rank-sum test. Benjamini-Hochberg correction for multiple
14
testing was used for the RNA sequencing as described in the previous paper (Trapnell et al
2010). p<0.001 and p<0.05 were considered to be significant as specified in the text.
15
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Details of Antibodies used for immunostaining
Antibody
Company
Catalog No.
Dilution
-catenin
BD transduction
610153
1:1000
Foxa2 (M20)
Santa Cruz
Biotechnology,
INC.
sc-6554
1:1500
Cyclin
D1
(Clone SP4)
Keratin 14
p63
Thermo Scientific
RM-9104-S
1:100
Covance
Santa Cruz
Biotechnology,
INC.
Cell Signaling
PRB-155P
sc-8431
1:200
1:200
2230
1:400
Novocastra
Laboratories Ltd.
Upstate (Millipore)
BD transduction
Upstate (Millipore)
Novocastra
Laboratories Ltd.
Upstate (Millipore)
--
1:300
06-570
610153
07-633
NCL-Ki67p
06-570
Lef-1
(C12A5)
NCL-Ki67p
pH3 (Ser10)
-catenin
Foxa2
Ki67
pH3 (Ser10)
19
Other Kits
Used
Vectastain ABC
kit
Vectastain ABC
kit, TSA
PerkinElmer,
INC.
Vectastain ABC
kit
-Vectastain ABC
kit
Usage
Vectastain ABC
kit, TSA
PerkinElmer,
INC.
--
IHC
1:300
1:100
1:50
1:100
----
IHC
IF
IF
IF
1:100
--
IF
IHC
IHC
IHC
IHC
IHC
IHC
Details of Antibodies used for Western Blotting
Antibody
Company
Catalog No.
Dilution
-catenin
BD transduction
610153
1:500
Foxa2 (M20)
Santa Cruz
Biotechnology,
INC.
sc-6554
1:600 diluted
with Toyobo
Can Get signal
NKB-101
1:1000 diluted
with Toyobo
Can Get signal
NKB-101
1:2000
Cyclin D1 (Clone Thermo Scientific
SP4)
RM-9104-S
-tubulin
B-5-1-2)
010M4813
(clone
Sigma-Aldrich
20
Detection
Reagents
Amersham ECL
Plus
Chemi-LumiOne
Nacalai Tesque
Amersham ECL
Plus
Amersham ECL
Plus