FLAG-PAWS1 HA-SMAD1 BMP GFP-SMAD4 +-+++--- ----++++ +++--+++ +--+++-- IP: GFP IB: HA IB: FLAG IB:...
-
Upload
osborn-shepherd -
Category
Documents
-
view
254 -
download
0
description
Transcript of FLAG-PAWS1 HA-SMAD1 BMP GFP-SMAD4 +-+++--- ----++++ +++--+++ +--+++-- IP: GFP IB: HA IB: FLAG IB:...
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