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ACCEPTED MANUSCRIPT Copyright 2016 The Author(s). This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the archiving policy of Portland Press Limited (http://www.portlandpresspublishing.com/content/open- access-policy#Archiving Policy). All other rights reserved. Cite as Biochemical Journal (2016) DOI: 10.1042/BCJ20160137 BIOCHEMICAL JOURNAL ACCEPTED MANUSCRIPT 10.1042/BCJ20160137 . Please cite using the DOI 10.1042/BCJ20160137 http://dx.doi.org/ up-to-date version is available at encouraged to use the Version of Record that, when published, will replace this version. The most this is an Accepted Manuscript, not the final Version of Record. You are
).
http://www.portlandpresspublishing.com/content/open-access-policy#Archiving Archiving Policy of Portland Press ( which the article is published. Archiving of non-open access articles is permitted in accordance with the Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under in vivo in vitro P P P P P P P P P P Figure 1. A μM 0 0.0005
0.001 0.0015
0.002 0.0025
0.003 0.0035
0.004 0.0045
0.005 0 50 100 Insulin secre on p
p rotein
(n g/ μ g) H 2 O 2 Control siRNA SCGN siRNA B * ** * 0 0.0005 0.001
0.0015 0.002
0.0025 0.003
0.0035 0 2.8 16.8 Insulin secre on p
p rotein
(n g/ μ g) Glucose
Control siRNA SCGN siRNA *** ***
*** mM tubulin CTL SCGN
siRNA An - -
An -SCGN CTL
SCGN siRNA
An - -tubulin An -SCGN
CTL SCGN
siRNA An - -tubulin An -SCGN C 0 1 2 3 4 5 6 7 8 9 10 3.3
16.8 In su lin r e le ase p e r isle ts (n g/ isle t) Control siRNA SCGN siRNA * Glucose mM B 0 16.8 CTL SCGN
CTL SCGN
An - -tubulin An -SCGN 5.4 30
siRNA CTL
SCGN CTL
SCGN Glucose
mM A 0 0.0005 0.001
0.0015 0.002
0.0025 0.003
0.0035 5.4
30 Insulin secre on p
p rotein
(n g/ g) Control siRNA SCGN siRNA KCl mM
0.0002 0.0004
0.0006 0.0008
0.001 0.0012
0 16.8
Insulin secre on p er total p rotein (n g/ g) Control siRNA SCGN siRNA Glucose mM
Figure 2. 0 0.0005 0.001 0.0015
0.002 0.0025
0.003 0 10 20 30 40 Ac cu m u late d in su lin se cr e on per tota l protei n (ng/ g) Time, min Control siRNA SCGN siRNA ***
* 0 SCGN siRNA 16.8 Control siRNA Glucose (mM)
C CTL
SCGN An - -tubulin An -SCGN siRNA
N N N PM IgG SCGN SCGN
IgG SCGN IgG SCGN Input Output IP IgG SCGN A C -ac n IP 58 46 30 25 - -ac n -SCGN - -ac n
-SCGN B Figure 3. AVFPSIVGRPR 599.8804(2+) mass
-0 50 100 150 200
250 300
350 400
450 500
550 600
650 700
750 800
850 900
950 1000
1050 1100
1150 % 0 100 5.86e3
881.5911 120.0866
70.0696 60.0574
72.0852 864.5797
567.3710 143.1257
272.1878 185.1036
245.1703 212.1519
468.2967 441.2512
280.1889 428.2893
314.1694 369.2815
397.2852 485.3235
524.3616 725.4980
584.3970 680.4709
628.4256 784.5338
821.5545 1198.7672 1028.6678 882.5065
1011.6394 927.6158
1029.5737 1042.6803 1043.5668 1167.6589 (P60710) Ac n, cytoplasmic 1 (Beta ac n) ACTB_Mouse Mass: 41710 Score: 99 Queries matched: 3 An body An body
Immunopre cipitated IP IgG
SCGN IgG SCGN
Glucose 0 16.8 mM - -ac n
-SCGN A B F-ac n Merge
SCGN 100
0 Ampli ed
- -ac n -SCGN
H 2 O 2 (μM)
C Figure 4. 0 5
15 20 25 30 35 40 1 2 3 4 5 6 7 8
ve in te n sit y ra o of S C GN (%) Frac on no. DMSO
Ionomycin 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 Rela ve in te n sit y ra o of ac n (%) Frac on no. DMSO
Ionomycin An -SCGN
An - -ac n DMSO
Ionomycin 1 2 3 4 5 6 7 8 Top frac on no. Bo om 1 2 3 4 5 6 7 8 Top frac on no. Bo om DMSO
Ionomycin IgG
SCGN IgG SCGN
Input Glucose 0 16.8 0 16.8 mM An body IgG
SCGN IgG SCGN
Output An body
80 90 100 110 120
130 140
150 160
170 180
0 16.8
-a c n/SC G N ra o (% ) mM Glucose *
Control SCGN
An - -ac n G-ac n F - ac n
siRNA G-ac n F-ac n An - -ac n An -SCGN
Total ac n Control SCGN A B
0.2 0.4
0.6 0.8
1 1.2
F- ac n to G -ac
n ra o (a
rbi tra
ry uni
ts ) Control siRNA SCGN siRNA * DAPI siRNA F-ac n
Merge SCGN
Ampli ed Control
0 100 Control SCGN 0
SCGN siRNA
Figure 5. H 2 O 2 (μM) Control siRNA SCGN siRNA A C
siRNA Merge
SCGN Control
0 100 Control SCGN 0
Paxillin An -E-Cadherin An -N-Cadherin siRNA CTL SCGN B An -SCGN
An -tubulin Figure 6. H 2
2 (μM)
Figure 7. An -p-ERK An -ERK An -p-AKT An -AKT An -SCGN
An -tubulin An -p-paxillin An -paxillin 0 5 15 30 min 0 5 15 30 H 2 O 2 An -p-FAK An -FAK Control
SCGN siRNA
0 2.8 16.8 30 Glucose
mM 0 2.8 16.8 30 An -SCGN An -p-FAK An -FAK An -p-paxillin An -paxillin An -p-ERK An -ERK An -p-AKT An -AKT Control
SCGN siRNA
0 2.8 16.8 30 Glucose
mM 0 2.8 16.8 30 0 50
150 200
250 300
0 2.8 16.8 30 Ph o sp h o -:To tal FAK (% ) Control siRNA SCGN siRNA 0 100 200 300
400 500
600 0 2.8 16.8 30 P h os pho- :T otal P axi lli n (%) Control siRNA SCGN siRNA 0 50 100 150
200 250
300 0 2.8 16.8 30 Ph o sp h o -:To tal Akt (% ) Control siRNA SCGN siRNA 0 50 100 150
200 250
300 0 2.8 16.8 30 Phospho-:Total E R K (%) Control siRNA SCGN siRNA * * ** ** * * * ***
****** Control
0 20 50 100 H 2 O 2 M SCGN An -SCGN
0 20 50 100 An -p-FAK An -FAK siRNA
An -p-paxillin An -paxillin An -p-ERK An -ERK
An -p-AKT An -AKT
Control 0 20 50 100 H 2
2 M SCGN 0 20 50 100 siRNA
0 50 100 150 200
250 300
350 400
0 20 50 100 Ph o sp h o -:To tal FAK (% ) Control siRNA SCGN siRNA 0 50 100 150
200 250
0 20 50 100 Ph o sp h o -:To tal Paxillin (% ) Control siRNA SCGN siRNA 0 100 200 300
400 500
600 0 20 50 100 Ph o sp h o -:To tal Akt (% ) Control siRNA SCGN siRNA 0 200 400 600
800 1000
1200 1400
1600 1800
0 20 50 100 P h os pho- :T o ta l E R K (%) Control siRNA SCGN siRNA ** * * * * * * *** *** C A B Control
SCGN siRNA
Glucose Glucose
Glucose Glucose
mM mM mM mM H 2 O 2 H 2 O 2 H 2 O 2 H 2 O 2 M M M M Control SCGN DMSO
Glucose Latrunculin B - + - + - + - + Control SCGN An -p-ERK An -ERK
An -p-FAK An -FAK
An -SCGN siRNA
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