FLAG-PAWS1HA-SMAD1

BMPGFP-SMAD4

+- +++-- -- - - - +++ +

++ +- - + +++ --+ + +--

IP: G

FP

IB: HA

IB: FLAG

IB: P-SMAD1 S206

IB: GFP

IB: P-SMAD1 S206

IB: HA

IB: FLAG

IB: P-SMAD1

IB: GFP

Inpu

t

Vogt et al. Figure S1

A

IB: SMAD1

Inpu

t IP: SMAD1

- TGFß BMPIgG

D

+---+

GFPGFP-SMAD1GFP-SMAD5GFP-SMAD8FLAG-PAWS1

-+--+

--+-+

---++

IB: PAWS1

IB: PAWS1

IB: GFP

IB: GFP

IP-F

LAG

inpu

t

HEK293

B

+----+

FLAGFLAG-SMAD1FLAG-SMAD1 MH1FLAG-SMAD1 LinkerFLAG-SMAD1 MH2HA-PAWS1

-+---+

--+--+

---+-+

IB: HA-PAWS1

IB: FLAGIP-F

LAG

inpu

t

HEK293

IB: FLAG

IB: HA-PAWS1

----++

C

A

MD

A-M

B-2

31

U2O

S

HE

K 2

93

RK

O

HaC

aT

HeL

a

MC

F7

PC3

KB

M7

IB: PAWS1 low

IB: PAWS1 high

IB: SMAD1

IB: GAPDH

B

IB: PAWS1 low

IB: PAWS1 high

IB: SMAD1

IB: GAPDH

PAWS1

PAWS1

Bra

inH

eart

Lun

gK

idne

yL

iver

Sple

enT

hym

usPa

ncre

asA

dipo

se ti

ssue

Mus

cle

Test

isE

pidi

dym

isO

vari

es

Vogt et al. Figure S2

32P-Autorad

LDN-193189 (µM) 0.00

00.

001

0.00

30.

010

0.03

00.

100

0.30

01.

000

3.00

010

.00

ALK2

Coomassie

0.00

00.

001

0.00

30.

010

0.03

00.

100

0.30

01.

000

3.00

010

.00

ALK6

Vogt et al. Figure S3

Phospho-PAWS1

R P A V A S S V S E E Y F E V R0

20406080

100120

32P

(cpm

) Peak1

R P A V A S S V S E E Y F E V R0

20406080

100120

32P

(cpm

) Peak2

BMPR1 + GST-PAWS1(S610A)

Vogt et al. Figure S4

A

B

C

0 16 32 48 64 80 96 1121281441601761922082242400

500

1000

1500

2000

2500

3000

3500

0102030405060708090

32P

radi

oact

ivity

/cpm

Time (min)

perc

enta

ge a

ceto

nitr

ile

P1P2

662.6184 +2P  

953.4414 +P

0102030405060708090

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160 180 200 220Time (min)

perc

enta

ge a

ceto

nitr

ile

32P

radi

oact

ivity

/ cp

m 32PACN %

Vogt et al. Figure S5

IP: PAWS1; IB: PAWS1

IB: P-SMAD1

IB: SMAD1

IB: GAPDH

IP: PAWS1;IB: P-PAWS1 S610

BMP for min. 0 15 45 120 240 480

BMPR2

0.0

0.5

1.0

1.5

2.0

fold

indu

ctio

n up

on B

MP

PC3 ControlPC3 PAWS1PC3 PAWS1 S610A

PC3 Control

PC3 PAWS1

PC3 PAWS1 S610A

BxPC3

- BMP 2 BMP 4/70

1

2

3ASNSNEDD9

fold

cha

nge

Vogt et al. Figure S6

A B

P<0.001 P<0.001

TGFBR2

0.0

0.5

1.0

1.5

2.0

2.5

fold

cha

nge

TSC22D

0.0

0.5

1.0

1.5

fold

cha

nge

unstimulatedBMP-2

Vogt et al. Figure S7

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log

fold

cha

nge

Change in expression PAWS1+BMP/control

genes below threshold

genes above threshold

Effect of Paws1 +BMPfold change fold change

TGFBI 8.53 4.73FST 3.02 3.27BMP6 2.96 3.23TGFBR2 2.61 2.40PDGFA 2.60 1.84MECOM 1.74 1.85INHBA -1.91 -1.65MYC -2.04 -1.86FOS -2.10SNAI1 -2.13 -2.06IGFBP3 -2.17 -1.93MSX2 -2.17 -1.86IL6 -2.38DLX2 -2.47 -2.97SERPINE1 -3.69 -6.04TSC22D1 -4.05 -3.73AR -7.74 -15.62CDKN1A 1.79HEY1 -1.86INHA -2.17

Effect of Paws1

Effect of Paws1 + BMPfold change fold change

AIPL1 9.44 3.32GSC 9.05IL10 8.55MYOD1 3.18AGT 2.61 -2.36BMPER 2.24 1.97CHRD -2.01SHH -2.68ACVRL1 -2.76BMP3 -3.54 5.77BMP7 -4.39CER1 -7.25 -3.51GDF6 -11.71 -6.88BDNF -4.07COL3A1 -2.78

Effect of Paws1Low expression genes

PC3-control

PC3-PAWS1

PC3-control

PC3-PAWS1

ID1

0

10

20

30

40

fold

cha

nge

PC3-control

PC3-PAWS1

P<0.0001

ID1

TGFBR2

TSC22D

A B

C

P<0.0001

P=0.0007P=0.0029