Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are
encouraged to use the Version of Record that, when published, will replace this version. The most
up-to-date version is available at http://dx.doi.org/10.1042/BCJ20160274. Please cite using the DOI
10.1042/BCJ20160274
BIOCHEMICAL JOURNAL
ACCEPTED MANUSCRIPT
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Cite as Biochemical Journal (2016) DOI: 10.1042/BCJ20160274
Copyright 2016 The Author(s).
This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the
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ACCEPTED MANUSCRIPT
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Figure 1
C
A
-
+
EBP1
neomycin
GST
PLCG1-PH GRP1-PH
10
EBP1
11
4
4
12
11
11
12
12
13
5
6
14
6
15
B
D
S1P
LPA 1
9
LPC 2
10
PtdIns(3,4)P2
PtdIns 3
11
PtdIns(3,5)P2
PtdIns(3)P 4
12
PtdIns(4,5)P2
PtdIns(4)P 5
13
PtdIns(3,4,5)P3
PtdIns(5)P 6
14
PA
PE 7
15
PS
7
E
300
Densitometry ratios
4
3
2
1
3P
4P
5P
34P2
35P2
45P2
PIP3
PA
PS
3P
4P
5P
34P2 35P2 45P2
PIP3
PA
PS
F
mM NaCl
150
5
0
Blank
400
1,6
Densitometry ratios (300/150)
PC 8
6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
Figure 2
A
B
K66
K65
K68
EBP1
355 KALLQSSAS-RKTQKKKKKKASKT
C Term
362
SAP30L 77
HKNFIQSVR-N-KRKRK-TS
Pf1
103 C-------TVR---RKKR-EQKKE
ING2
256 CRGDNEKTMDKSTEKTKKD--RRSR
BASP1
1
MGGKLS-KKKKGYNVN
C
N-term motif:
N-WT: 65KKEKEMKK72
N-K3A: 65AAEAEMKK72
C-term motif:
C-WT: 364RKTQKKKKKK373
C-K4A: 364RKTQKAAAAK373
FL GST
1
394
N-term GST
1
351
C-term GST
1
352
394
D
N-term EBP1
N-WT
C-term EBP1
C-WT
N-K3A
Full Length EBP1
WT
C-K4A
N-K3A/C-K4A
10
11
11
4
5
6
11
4
12
4
5
13
5
6
14
6
Figure 3
A
B
Merge
NPM
EBP1
putative NoLS: 357-85 LLQSSASRKTQKKKKKKASKTAENATSGE
C-term PBR:
364-73
RKTQKKKKKK
C
C-K4A
N-K3A
N-K3A/C-K4A
Merge
NPM
EGFP
WT
D
120
E
100
- WT
80
pattern #1
%
60
EGFP-EBP1
pattern #2
pattern #3
40
EGFP
20
D-tubulin
0
WT
Figure 4
A
NLS-EGFP
+
-
+
Merge
Nucleolin
EGFP
-
NLS-EGFP-GRP1-PH
B
C
pmol
PtdIns
PtdIns(3)P
PtdIns(4)P
PtdIns(5)P
PtdIns(3,4)P2
PtdIns(3,5)P2
PtdIns(4,5)P2
PtdIns(3,4,5)P3
%
-
D
E
+
Nucleolin
PtdIns(3,4,5)P3
Merge
NPM
p110E
Merge
Figure 5
A
PtdIns(3,4,5)P3
EBP1
B
-
+
anti-EBP1
Merge
C
PtdIns(3,4)P2
250
150
100
PtdIns(3,5)P2
75
PtdIns(3)P
PtdIns(4,5)P2
50
PtdIns(4)P
PtdIns(3,4,5)P3
PtdIns(5)P
37
25
EBP1
Figure 6
B
A
N [ppm]
G393
G350*
15
G384
T382
S363
T377
115
T366
S375
S360
S383
E389
L346*
K365
K368
Q367
K372
K376
K373
L387
K355 L357
L358
A356
D394
A362
A374
SSP plot
V347*
R364
A381
A392
E391 E388
E385
120
N390
125
R349*
D345*
S351*
T386
S361
Q359
E379
N380
130
A378
1
8.5
8.0
7.5
7.0
6.5
1
H [ppm]
D
C
15 N [ppm]
G393
G350*
G384
T382
S363
T377
115
T366
S375
S360
S383
K376
K373
L387
K365
K368
Q367
K372
L358
R364
intensity C16
A356
A381
A392
E389
E391 E388
K355 L357
120
N390
T386
S361
Q359
E379
E385
L346*
V347*
125
R349*
D345*
S351*
N380
D394
A362
A374
A378
130
1
E
8.0
7.5
7.0
6.5
H [ppm]
F
intensity C8
8.5
1
Figure 7
A
WT
364-RKTQKKKKKK-373
K372N 364-RKTQKKKKNK-373
B
NPM
Merge
K372N
EGFP
D
C
K372N
1,2
PI(3,4)P2
PI(4,5)P2
PI(3,4,5)P3
PA
PS
Densitometry ratio K372N/WT
WT
1
0,8
0,6
0,4
0,2
0
3P
-0,2
4P
5P
34P2
35P2
45P2 345P3
PA
PS