2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab...
Transcript of 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab...
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2BME:2-mercaptoethanol
Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug)
Ephrin-B2 Ab + Rc Ephrin B2 (5 ug)
Ephrin-B2 Oligomers
Ephrin-B2 Monomer
1236
480
kDa
242
SupplementaryFigure1
Ephrin-B2Dimer
Nature Medicine: doi:10.1038/nm.4419
SupplementaryFigure2
Nature Medicine: doi:10.1038/nm.4419
SupplementaryFigure3
Healthy fibroblasts
IPF fibroblasts
a
80
40
kDa
Ephrinb2floxed552bpWildtype450bp
b
Lungfibroblasts Lungmacrophages
80
40
EphrinB2
40Beta-ac=n
kDa
kDa
kDa
PBS Bleomycin
c
d
AlphaSMA
TypeICollagen
Beta-Ac=n
150
kDa
40
kDa
40
kDa
40 GAPDH
EphrinB2
Cre408bp
Nature Medicine: doi:10.1038/nm.4419
EphrinB2
PBS Bleomycin 1 2 3 4
PBS Bleomycin
1 2 3 4 1 2 3 4
RecombinantEphrin-b2
SupplementaryFigure4
1 2 3 4 5 6 7 8
80
40
kDa
a
b
sEphrinB2
40
GAPDH
kDa
40GAPDH
80
Nature Medicine: doi:10.1038/nm.4419
Native-PAGE
- SDS + 2ME
Ephrin-B2Oligomer
+ - +
Ephrin-B2Monomer
Ephrin-B2Oligomer
Ephrin-B2Dimer
SupplementaryFigure5
ControlBAL
BleoBAL
1236
720
kDa
NaCve-PAGE
480
242
a
b
AlphaSMA
TypeICollagen
Beta-ac=n
kDa
kDa
kDa
kDa
kDa
1236
480
kDa
242
ControlBAL
BleoBAL
37
150
37
kDa
37
150
AlphaSMA
TypeICollagen
Beta-ac=n37
c
d
1048
1048
Nature Medicine: doi:10.1038/nm.4419
SupplementaryFigure6
Lowexposure
Higherexposure
NT siRNA
ADAM10 siRNA
TGF-β - - + +
AlphaSMA
GAPDH
a
37
kDa
37
kDa
Lowerexposure
HigherExposure
AlphaSMA
GAPDH37
kDa
kDa
c
b
GFP WT Δ197218 ΔJuxta Δ182/194 GFP WT Δ 197218 Δ Juxta Δ182/194
37
TGF-β - + - + - + - + - + - + - + - + - + - +
GAPDH37
kDa
HA
Lowerexposure HigherExposure
80
40
kDa
HA
kDa
80
40
TGF-β - + GFP
- + GFP - +
WT - + WT
ADAM10 siRNA
- +
Nature Medicine: doi:10.1038/nm.4419
SupplementaryFigure6
AlphaSMA
d
kDa
GAPDH
GAPDH
AlphaSMA
e
-+-+-+-+-+
40
kDa
kDa
Δ18
2/19
4
Eph
rin-B
2-W
T
Δ19
7/21
8
GFP
TGF-β
- - + + - - + + TGF-β Δ18
2/19
4
Eph
rin-B
2-W
T
Δ19
7/21
8
GFP
- + + - - + + TGF-β -
Nature Medicine: doi:10.1038/nm.4419
Bleomycin Day 7
Day 14
Day 0
Day 1
Day 3
SupplementaryFigure7
80
kDa
40
kDa
ADAM10
Beta-acCn
a
b
PBS BLM BLM + GI25
40
kDa
AlphaSMA
Beta-acCn40
PBS
Nature Medicine: doi:10.1038/nm.4419
SupplementaryFigure8
sEphrinB2
Control BAL IPF BAL 1 2 3 4 5 6 1 2 3 4 5 6
37
kDa
60
RecombinantEphrin-b2
Nature Medicine: doi:10.1038/nm.4419
Supplementary Figure Legends
Supplementary Figure 1. Effect of denaturing and reducing conditions on sEphrin-B2
oligomerization. BAL samples were run in triplicates on non denaturing gels (native
PAGE) with or without SDS or SDS + 2-mercaptoethanol. Individual membranes were
then incubated with a) ephrin-B2 antibody, b) Ephrin-B2 antibody preincubated with 1 µg
of recombinant mouse ephrin-B2 or c) Ephrin-B2 antibody pre-incubated with 5 µg of
recombinant mouse ephrin-B2.
Supplementary Figure 2 siRNA transfection efficiency. Human lung fibroblasts were
transfected with non-targeting control siRNA or ON-TARGETplus siRNA against
MMP1, MMP2, MMP7, MMP14, ADAM9, ADAM10, ADAM12, ADAM17, ADAM19,
EPHB3 and EPHB4. ON-TARGETplus siRNA uses a validated pool of 4 individual
siRNAs per target gene. Cells were transfected with either 10 nM or 50 nM siRNA and
gene expression levels tested by qPCR 48 hours post-transfection. Data are presented as
mean ± SD. P < 0.05, Student’s t-test.
Supplementary Figure 3 Uncropped blot images from Figure 1. (a). Uncut blots for
Figure 1b. (b) Uncut gels for Figure 1d. (c) Uncut blots for Figure 1e. (d). Uncut blots
for Figure 1i.
Supplementary Figure 4. Uncropped blot images from Figure 2. (a) Uncut blots for
Figure 2a. (b) Uncut blots for Figure 2b.
Supplementary Figure 5. Uncropped blot images from Figure 3. (a). Uncut blots for
Figure 3h. (b) Uncut gels for Figure 3i. (c) Uncut blots for Figure 3l. (d). Uncut blots for
Figure 3p.
Nature Medicine: doi:10.1038/nm.4419
Supplementary Figure 6. Uncropped blot images from Figure 4. (a). Uncut blots for
Figure 4h. (b) Uncut gels for Figure 4j. (c) Uncut blots for Figure 4n. (d). Uncut blots for
Figure 4p. (e) Uncut blots for Figure 4s.
Supplementary Figure 7. Uncropped blot images from Figure 5. (a). Uncut blots for
Figure 5a. (b) Uncut gels for Figure 5d.
Supplementary Figure 8. Uncropped blot images from Figure 6. (a). Uncut blots for
Figure 6i.
Nature Medicine: doi:10.1038/nm.4419
Supplementary Table 1: IPF and Control Subject Characteristics. IPF (n = 30) Controls (n = 30) Age (yrs) 68.5 (7.0) 68.3 (7.0) Sex (M/F) 21/9 21/9 Smoking (current/ever/never)
0/26/4 0/26/4
FVC predicted (%) 66.8 (14.9) N/A DLCO [Hb] predicted (%) 44.0 (18.2) N/A Data are presented as mean +/- SD as appropriate. N/A refers to data that was not available or collected. Pulmonary function data (FVC/DLCO [Hb] – the long forms of this should be defined) was performed within 15 days of plasma collection and was available on 27/30 subjects.
Nature Medicine: doi:10.1038/nm.4419
Supplementary Table 2
Gene Species Sequence (5’ to 3’)
Collagen type I α1 chain (col1α1)
Human F: GAACGCGTGTCATCCCTTGT R: GAACGAGGTAGTCTTTCAGCAACA
α-smooth muscle actin (α-SMA)
Human F: TTCAATGTCCCAGCCATGTA R: GAAGGAATAGCCACGCTCAG
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Human F: CCCATGTTCGTCATGGGTGT R: TGGTCATGAGTCCTTCCACGATA
Ephrin A1 Human
F:CGGAATGAGGACTACACCATACATGTGCAGC R:AAGCAGCGGTCTTCATGCTGGTGGATGGGTT
Ephrin A2 Human F: CTACACGGTGGAGGTGAGCA R: ACAGCATTGGGAGGCGTGGCA
Ephrin A3 Human F: ACTACATCTCCACGCCCAC R: TCCCGCTGATGCTCTTCTC
Ephrin A4 Human F: TTGTACATGGTGGACTGGCC R: AGAACTCTCTGGAGTGGGCACC
Ephrin A5 Human F: CCAGAAGATAAGACTGAGCGC R: CCATTATCTGGGATTGCAGAGG
Ephrin B1 Human F: CCAATGCTGTGACGCCTGAG R: CGAACAATGCCACCTTGGAGTTG
Ephrin B2 Human F: GGAAGAAGTTCGACAACAAGTCC R: TTCAGCAAGAGGACCACCAGCGT
Ephrin B3 Human F: TGGAGCCTGGGAAGGAGAACC R: CAGGGTGGCGACTCTCCGAAG
EphB1 Human F: TTCACTTCAGCCAGCGAC R: TTTCCCTCCTCTCCTTCCC
Nature Medicine: doi:10.1038/nm.4419
EphB2 Human F: ATGGCGCCCCTCTCCTCTGGCATCA R: ACCGCTTGGTTCTTCCCGTG
EphB3 Human F: GCATCGCCTCCACAGTGACC R: ACGAAGACAAGCCCAGCTGTA
EphB4 Human F: TTCGGCCAGGAACATCACAG R: CCGATGAGATACTGTCCGTG
EphB6 Human F: TACCTGTCCAGCTTTGCC R: TCTCCATAACTCATCACTTCCC
MMP1 Human F: GGTCTCTGAGGGTCAAGCAG R: AGTTCATGAGCTGCAACACG
MMP2 Human F: ACAGCAGGTCTCAGCCTCAT R: TGAAGCCAAGCGGTCTAAGT
MMP3 Human F: GCAGTTTGCTCAGCCTATCC R: GAGTGTCGGAGTCCAGCTTC
MMP7 Human F: GAGTGCCAGATGTTGCAGAA R: AAATGCAGGGGGATCTCTTT
MMP8 Human F: TCTGCAAGGTTATCCCAAGG R: CTTGCTGGAAAACTGCATCA
MMP9 Human F: TCTGCAAGGTTATCCCAAGG R: CTTGCTGGAAAACTGCATCA
MT1-MMP/MMP14
Human F: CAGAGAAGGCACACAAACGA R: CACTGGTGAGACAGGCTTGA
ADAM8 Human F: ACCCTTCCCAGTTCCTGTCT R: CTCCTTGCTTCCTCTGGATG
ADAM9 Human F: GTTCCTGTGGAGCAAAGAGC R: CCAGCGTCCACCAACTTATT
ADAM10 Human F: AGCAACATCTGGGGACAAAC R: CCCAGGTTTCAGTTTGCATT
ADAM12 Human F: GCCTCTGTCTTCTGGACTGG R: TGGAAGGCAAACCAAGATTC
ADAM15 Human F: AGCCTCAAAAAGGTGCTTCA R: CCCTGGTAGCAGCAGTTCTC
Nature Medicine: doi:10.1038/nm.4419
ADAM17/TACE Human F: TGAGGGCAGTTAACCAAACC R: ATACACCCACACACCCCACT
ADAM19 Human F: CTGAAGGCTGTGGGAAGAAG R: ACCACAGGACCCACACTCTC
ADAM28 Human F: TTCAGTCAGGGGGATCAAAG R: TAGTTTGGTGGCAGGTAGGG
Collagen type I α1 chain (col1α1)
Mouse
F: CCGTGCTTCTCAGAACATCA R: GAGCAGCCATCGACTAGGAC
α-smooth muscle actin (α-SMA)
Mouse
F: CTGACAGAGGCACCACTGAA R: CATCTCCAGAGTCCAGCACA
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Mouse
F: AACTTTGGCATTGTGGAAGG R: GGATGCAGGGATGATGTTCT
Ephrin A1
Mouse F: CCCACATTACGAGGACGACT R: GTGAAGCGCTGGAATTTCTC
Ephrin A2
Mouse F: CTCCGAGACAAAATCCTTGC R: CTCCGAGACAAAATCCTTGC
Ephrin A3
Mouse F: CCTGCACTGGAAGTGTCTGA R: GGGGATTCTCTCCCTCAAAG
Ephrin A4
Mouse F: GCAAAGTCGGAGGATCTGAG R: GCCCTCAGTGTCTCAAGGAG
Ephrin A5
Mouse F: TCCAGAGGGGTGACTACCAC R: CTTGAACCCTTTGGACGTGT
Ephrin B1
Mouse F: ATCGCAAGCATACACAGCAG R: CTGGGCCTTCAAACCTTGTA
Ephrin B2
Mouse F: ACAGGTGGGAGGTGACTGAC R: GCTGCGCTTTTTATTTCCAG
Ephrin B3
Mouse F: TGTTCTCCCCACCTAACCAG R: GCACACTAAAGAGCGGGAAG
EphB1
Mouse F: GATTTTGGGGGAAGGTTGAT R: ATGGTTTCTGGGAACACTGC
EphB2
Mouse F: ACTATGGCGGCTGTATGTCC R: GCACATCCACTTCTTCAGCA
Nature Medicine: doi:10.1038/nm.4419
EphB3
Mouse F: CAAGGCTGGCTTCTACTTGG R: GAGACCTCCACAGCGTTAGC
EphB4
Mouse F: AATGTCACCACTGACCGTGA R: TCAGGAAACGAACACTGCTG
EphB6
Mouse F: CGAGAGGGCCAGTTCAGTAG R: GCGAAGCAAGGAACTTGAAC
Genotyping primers
Cre mice
Mouse
Cre-1s: GCATTACCGGTCGATGCAACGAGTGATG
Cre mice
Mouse
Cre-2as: GAGTGACGAACCTGGTCGAAATCAGTCG
Ephrin-B2 flox mice
Mouse EPHB2_1s:
TCATTTCCCAACCACCGCCAGAAA
Ephrin-B2 flox mice
Mouse EPHB2_2as:
AGATACCACGCCAGGAGAGCAAAT
Supplementary Table 2. Primer pairs used for quantitative real-time PCR. The table shows the specific gene detected, the species (mouse or human) and the corresponding sequence for forward (F) and reverse (R) primers.
Nature Medicine: doi:10.1038/nm.4419