16
Control Control + SDS Control + SDS + 2BME Control Control + SDS Control + SDS + 2BME Control Control + SDS Control + SDS + 2BME 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 Supplementary Figure 1 Ephrin-B2 Dimer Nature Medicine: doi:10.1038/nm.4419

2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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Page 1: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

Contro

l Con

trol +

SDS

Contro

l + S

DS +

2BM

E

Contro

l Con

trol +

SDS

Contro

l + S

DS +

2BM

E

Contro

l Con

trol +

SDS

Contro

l + S

DS +

2BM

E

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

Page 2: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

SupplementaryFigure2

Nature Medicine: doi:10.1038/nm.4419

Page 3: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 4: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 5: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 6: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 7: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 8: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 9: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 10: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 11: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 12: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 13: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

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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

Page 15: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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

Page 16: 2BME:2-mercaptoethanol - Nature · BME S BME S 2BME:2-mercaptoethanol BME Ephrin-B2 Ab Ephrin-B2 Ab + Rc Ephrin B2 (1 ug) Ephrin-B2 Ab + Rc Ephrin B2 (5 ug) Ephrin-B2 Oligomers Ephrin-B2

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