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Saheli Sengupta, Uttama Rath, Changfu Yao, Michael Zavortink, Chao Wang, Jack Girton, Kristen M. Johansen, Jørgen Johansen
Digitor/dASCIZ Has Multiple Roles in Drosophila Development
www.plosone.org
www.plosone.org
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5.0116 0 Td
(CAB4211,)Tj
4.2747 0 Td
(RRID:)Tj
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6.3495 0 Td
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2.8573 0 Td
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3.4299 0 Td
(IIo)Tj
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Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 338.2866 568.6299 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
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(ser5)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 352.6866 564.2078 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(\(Covance,)Tj
4.2406 0 Td
(MMS-134R,)Tj
5.1023 0 Td
(RRID:AB_10119940\),)Tj
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(anti-HA)Tj
3.5717 0 Td
(\(Roche,)Tj
3.2881 0 Td
(11583816001,)Tj
5.675 0 Td
(RRID:AB_514505\),)Tj
7.9425 0 Td
(and)Tj
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-32.7852 -1.3039 Td
(#9701,)Tj
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(RRID:AB_331535\).)Tj
7.9427 0 Td
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(lamin)Tj
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Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 366.4062 536.5417 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
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(0)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 372.1889 538.1858 cm
BT
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1 TL
-0.004 Tc
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(mAb)Tj
2.228 0 Td
(HL1203)Tj
3.4299 0 Td
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1.7007 0 Td
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1.1792 0 Td
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(M.)Tj
1.3379 0 Td
(Paddy)Tj
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-35.5462 -1.2982 Td
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(Saumweber)Tj
4.8869 0 Td
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1.542 0 Td
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4.354 0 Td
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5.5558 0 Td
([)Tj
0.83 0.64 0.02 0 k
(16)Tj
0 g
(].)Tj
2.0806 0 Td
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1.7801 0 Td
(mouse)Tj
2.8573 0 Td
(anti-BrdU)Tj
4.337 0 Td
(mAb)Tj
2.228 0 Td
(\(RRID:)Tj
-34.6844 -1.2983 Td
(AB_2618097\))Tj
5.6466 0 Td
(and)Tj
1.7348 0 Td
(the)Tj
1.4683 0 Td
(mouse)Tj
2.8573 0 Td
(anti-)Tj
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(\r)Tj
/F9 1 Tf
(H2Av)Tj
4.955 0 Td
(mAb)Tj
2.2335 0 Td
(\(RRID:AB_2618077\))Tj
8.5323 0 Td
([)Tj
0.83 0.64 0.02 0 k
(17)Tj
0 g
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1.8538 0 Td
(were)Tj
2.1203 0 Td
(obtained)Tj
3.7304 0 Td
(from)Tj
-35.1323 -1.3039 Td
(the)Tj
1.4684 0 Td
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6.2985 0 Td
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3.1011 0 Td
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4.8755 0 Td
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ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 463.9748 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
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(Biochemical)Tj
5.5417 0 Td
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ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 447.0236 cm
BT
/F14 1 Tf
1 TL
-0.004 Tc
0 0 Td
[( )8( )65()35($\n)]TJ
4.575 0 Td
[( )16()6(.)]TJ
1.7292 0 Td
[()12()18()18()12()6()13()10()4()12(##)7()12()13(")]TJ
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6.7747 0 Td
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3.1918 0 Td
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(20)Tj
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1.5023 0 Td
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1.6214 0 Td
(150)Tj
1.6498 0 Td
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1.9332 0 Td
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2.5228 0 Td
(10)Tj
1.1736 0 Td
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1.9332 0 Td
(EDTA,)Tj
3.0444 0 Td
(1)Tj
-37.0599 -1.2982 Td
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1.9275 0 Td
(EGTA,)Tj
3.016 0 Td
(0.2%)Tj
2.1487 0 Td
(Triton)Tj
2.8233 0 Td
(X-100,)Tj
2.8686 0 Td
(0.2%)Tj
2.1487 0 Td
(NP-40,)Tj
3.0387 0 Td
(2)Tj
0.686 0 Td
(mM)Tj
1.9275 0 Td
(Na)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 417.6 419.3574 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 421.1149 421.0015 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(VO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 435.5716 419.3574 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 439.0866 421.0015 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4422 0 Td
(1)Tj
0.6916 0 Td
(mM)Tj
1.9275 0 Td
(PMSF,)Tj
2.8914 0 Td
(1.5)Tj
/F17 1 Tf
1.3946 0 Td
[()]TJ
/F9 1 Tf
(g/ml)Tj
2.5795 0 Td
(aprotinin)Tj
-33.834 -1.2982 Td
(as)Tj
1.0148 0 Td
(previously)Tj
4.3483 0 Td
(described)Tj
4.0535 0 Td
([)Tj
0.83 0.64 0.02 0 k
(18)Tj
0 g
(].)Tj
2.075 0 Td
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3.5546 0 Td
(were)Tj
2.126 0 Td
(separated)Tj
4.0081 0 Td
(by)Tj
1.1849 0 Td
(SDS-PAGE)Tj
4.7848 0 Td
(and)Tj
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(immunoblotted)Tj
-28.8791 -1.3039 Td
(according)Tj
4.1839 0 Td
(to)Tj
1.0261 0 Td
(standard)Tj
3.7247 0 Td
(procedures)Tj
4.6715 0 Td
([)Tj
0.83 0.64 0.02 0 k
(10)Tj
0 g
(].)Tj
2.0806 0 Td
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1.6214 0 Td
(these)Tj
2.2564 0 Td
(experiments)Tj
5.1419 0 Td
(we)Tj
1.3266 0 Td
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2.058 0 Td
(the)Tj
1.4683 0 Td
(Bio-Rad)Tj
3.5093 0 Td
(Mini)Tj
2.194 0 Td
(PRO-)Tj
-35.2627 -1.2983 Td
(TEAN)Tj
2.8176 0 Td
(III)Tj
1.2416 0 Td
(system,)Tj
3.1634 0 Td
(electroblotting)Tj
6.0491 0 Td
(to)Tj
1.0262 0 Td
(0.2)Tj
/F17 1 Tf
1.4003 0 Td
[()]TJ
/F9 1 Tf
(m)Tj
1.542 0 Td
(nitrocellulose,)Tj
5.845 0 Td
(and)Tj
1.729 0 Td
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2.3925 0 Td
(anti-mouse)Tj
4.7565 0 Td
(or)Tj
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(anti-rabbit)Tj
-33.063 -1.2983 Td
(HRP-conjugated)Tj
6.9278 0 Td
(secondary)Tj
4.2746 0 Td
(antibody)Tj
3.7644 0 Td
(\(Bio-Rad\))Tj
4.1839 0 Td
(\(1:3000\))Tj
3.5036 0 Td
(for)Tj
1.3889 0 Td
(visualization)Tj
5.2441 0 Td
(of)Tj
1.0148 0 Td
(primary)Tj
3.4582 0 Td
(antibody.)Tj
-33.7603 -1.3039 Td
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4.0138 0 Td
(labeling)Tj
3.3562 0 Td
(was)Tj
1.7008 0 Td
(visualized)Tj
4.1386 0 Td
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(chemiluminescent)Tj
7.5684 0 Td
(detection)Tj
3.9401 0 Td
(methods)Tj
3.685 0 Td
(\(SuperSignal)Tj
-30.7953 -1.2982 Td
(West)Tj
2.262 0 Td
(Pico)Tj
1.9673 0 Td
(Chemiluminescent)Tj
7.8009 0 Td
(Substrate,)Tj
4.1385 0 Td
(Pierce\).)Tj
3.2485 0 Td
(The)Tj
1.7745 0 Td
(immunoblots)Tj
5.6238 0 Td
(were)Tj
2.1146 0 Td
(digitized)Tj
3.668 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.6463 0 Td
(Che-)Tj
-35.6312 -1.2983 Td
(miDoc-It)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 237.4299 334.4314 cm
BT
/F18 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 244.9133 330.0094 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(TS2)Tj
1.7801 0 Td
(Imager)Tj
3.0727 0 Td
(\(UVP,LCC\))Tj
4.9833 0 Td
(and)Tj
1.7291 0 Td
(densitometric)Tj
5.7543 0 Td
(analysis)Tj
3.3279 0 Td
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1.7008 0 Td
(done)Tj
2.2223 0 Td
(using)Tj
2.3924 0 Td
(the)Tj
1.4684 0 Td
(built-in)Tj
-32.9213 -1.2982 Td
(software.)Tj
/F14 1 Tf
1.1962 -1.3039 Td
[()2()4()4(.)4()12(/)-13()]TJ
4.2519 0 Td
[()1(!)4()2()-9()12()6()2()22(#)]TJ
/F9 1 Tf
(.)Tj
5.125 0 Td
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1.627 0 Td
[()12()]TJ
1.0148 0 Td
[(0)-13()14(#)7()20()]TJ
/F9 1 Tf
2.0466 0 Td
(pull)Tj
1.7575 0 Td
(down)Tj
2.4831 0 Td
(assays)Tj
2.6306 0 Td
(of)Tj
1.0148 0 Td
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6.701 0 Td
(interactions,)Tj
5.1931 0 Td
(a)Tj
0.6576 0 Td
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-35.6992 -1.2983 Td
(letor)Tj
2.0636 0 Td
(cDNA)Tj
2.8005 0 Td
(fragment)Tj
3.8721 0 Td
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3.9288 0 Td
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(215474)Tj
3.5943 0 Td
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1.6952 0 Td
(subcloned)Tj
4.2916 0 Td
(in)Tj
1.0375 0 Td
(frame)Tj
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(into)Tj
1.8425 0 Td
(the)Tj
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1.4683 0 Td
[(\n)14(\)1 )]TJ
/F9 1 Tf
3.2485 0 Td
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-35.8806 -1.2983 Td
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1.3946 0 Td
(\(Novagen\))Tj
4.4673 0 Td
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4.5297 0 Td
(6x)Tj
1.1622 0 Td
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3.7474 0 Td
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(cDNA)Tj
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-35.8863 -1.3039 Td
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3.4809 0 Td
(144)Tj
1.6497 0 Td
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1.0204 0 Td
(376)Tj
1.6498 0 Td
(without)Tj
3.3278 0 Td
(the)Tj
1.4741 0 Td
(NH)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 341.0645 263.3385 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 344.6362 264.9826 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
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(-terminal)Tj
3.9798 0 Td
(zinc)Tj
1.8708 0 Td
(finger)Tj
2.5795 0 Td
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1.4683 0 Td
[($\n2&\r)]TJ
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4.3994 0 Td
(vector)Tj
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5.0002 0 Td
(Biosciences\))Tj
5.1477 0 Td
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1.9956 0 Td
(a)Tj
0.6519 0 Td
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3.7984 0 Td
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1.2643 0 Td
(a)Tj
0.652 0 Td
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3.1237 0 Td
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3.9798 0 Td
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6.5536 0 Td
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1.474 0 Td
(JIL-1)Tj
2.2506 0 Td
(kinase)Tj
2.7496 0 Td
([)Tj
0.83 0.64 0.02 0 k
(19)Tj
0 g
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0.83 0.64 0.02 0 k
(20)Tj
0 g
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3.033 0 Td
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6.0548 0 Td
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1.4683 0 Td
(first)Tj
1.8085 0 Td
(260)Tj
1.6441 0 Td
(amino)Tj
2.8006 0 Td
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-35.7445 -1.3039 Td
(residues)Tj
3.4809 0 Td
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1.7008 0 Td
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4.2916 0 Td
(in)Tj
1.0375 0 Td
(frame)Tj
2.5511 0 Td
(into)Tj
1.8425 0 Td
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1.4683 0 Td
[(\n)14(\)1 )]TJ
/F9 1 Tf
3.2541 0 Td
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2.9254 0 Td
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1.7801 0 Td
(tagged)Tj
2.8347 0 Td
(protein)Tj
3.1521 0 Td
(constructs)Tj
4.3426 0 Td
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-34.6617 -1.2983 Td
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4.0932 0 Td
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1.0375 0 Td
(BL21)Tj
2.2847 0 Td
(cells)Tj
1.9218 0 Td
(\(Novagen\).)Tj
4.6942 0 Td
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1.627 0 Td
(His)Tj
1.6157 0 Td
(pull-down)Tj
4.3767 0 Td
(assays,)Tj
2.8516 0 Td
(approximately)Tj
5.9754 0 Td
(10)Tj
/F17 1 Tf
1.1679 0 Td
[()]TJ
/F9 1 Tf
(g)Tj
1.1962 0 Td
(of)Tj
1.0148 0 Td
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-33.8567 -1.2982 Td
(letor)Tj
2.0636 0 Td
(or)Tj
1.0998 0 Td
(His-JIL-1)Tj
4.0025 0 Td
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3.3902 0 Td
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2.0012 0 Td
(Ni-NTA)Tj
3.6056 0 Td
(beads)Tj
2.4718 0 Td
(\(Qiagen\))Tj
3.7871 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1783 0 Td
(with)Tj
1.9955 0 Td
(10)Tj
/F17 1 Tf
1.1736 0 Td
[()]TJ
/F9 1 Tf
(g)Tj
1.1905 0 Td
(of)Tj
-34.8091 -1.3039 Td
(GST-Digitor)Tj
5.2837 0 Td
(in)Tj
1.0375 0 Td
(1X)Tj
1.3493 0 Td
(PBS)Tj
1.8312 0 Td
(\(pH)Tj
1.8481 0 Td
(7.4\))Tj
1.7348 0 Td
(for)Tj
1.3832 0 Td
(4)Tj
0.6973 0 Td
(hr)Tj
1.1226 0 Td
(at)Tj
0.9467 0 Td
(4C.)Tj
1.9786 0 Td
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1.7858 0 Td
(protein)Tj
3.1521 0 Td
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7.1433 0 Td
(beads)Tj
2.4718 0 Td
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-33.766 -1.2983 Td
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2.3754 0 Td
(a)Tj
0.6576 0 Td
(total)Tj
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(of)Tj
1.0148 0 Td
(5)Tj
0.6973 0 Td
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3.0104 0 Td
(of)Tj
1.0148 0 Td
(20)Tj
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(min)Tj
1.8538 0 Td
(each)Tj
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(\(2)Tj
1.0318 0 Td
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3.0161 0 Td
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1.9956 0 Td
(0.04%)Tj
2.6305 0 Td
(PBST,)Tj
2.6702 0 Td
(2)Tj
0.6973 0 Td
(washes)Tj
3.0104 0 Td
(with)Tj
1.9956 0 Td
(0.02%)Tj
-32.8759 -1.2983 Td
(PBST,)Tj
2.6702 0 Td
(and)Tj
1.7291 0 Td
(a)Tj
0.652 0 Td
(final)Tj
2.0069 0 Td
(wash)Tj
2.228 0 Td
(in)Tj
1.0318 0 Td
(1X)Tj
1.3493 0 Td
(PBS\))Tj
2.177 0 Td
(and)Tj
1.729 0 Td
(analyzed)Tj
3.702 0 Td
(by)Tj
1.1793 0 Td
(SDS-PAGE)Tj
4.7905 0 Td
(and)Tj
1.7291 0 Td
(immunoblotting)Tj
6.8485 0 Td
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-33.8227 -1.2982 Td
(anti-GST)Tj
3.9174 0 Td
(mAb)Tj
2.2337 0 Td
(8C7)Tj
1.8312 0 Td
([)Tj
0.83 0.64 0.02 0 k
(3)Tj
0 g
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1.6044 0 Td
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3.7984 0 Td
(for)Tj
1.3889 0 Td
(GST)Tj
2.0239 0 Td
(pulldown)Tj
4.0252 0 Td
(assays,)Tj
2.8516 0 Td
(GST-Digitor)Tj
5.2837 0 Td
(or)Tj
1.0942 0 Td
(GST)Tj
2.0239 0 Td
(alone)Tj
2.3811 0 Td
(was)Tj
-34.4576 -1.304 Td
(bound)Tj
2.8346 0 Td
(to)Tj
1.0261 0 Td
(glutathione)Tj
4.7622 0 Td
(agarose)Tj
3.2202 0 Td
(beads)Tj
2.4717 0 Td
(\(Sigma\))Tj
3.3618 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1783 0 Td
(with)Tj
1.9956 0 Td
(His-tagged)Tj
4.5807 0 Td
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(CCD)Tj
2.2847 0 Td
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ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 628.9511 cm
BT
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1 TL
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4.7764 0 Td
(confocal)Tj
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Q
q
1 j
1 J
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(imaging)Tj
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(700.)Tj
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-34.9793 -1.3039 Td
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2.1996 0 Td
(image)Tj
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(of)Tj
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(z-plane)Tj
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3.3562 0 Td
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1.0148 0 Td
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1.474 0 Td
(mitotic)Tj
3.1011 0 Td
(appa-)Tj
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(were)Tj
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(obtained)Tj
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(a)Tj
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7.1943 0 Td
(63X)Tj
1.8255 0 Td
(1.4)Tj
1.3946 0 Td
(NA)Tj
1.6441 0 Td
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 507.4582 cm
BT
/F0 1 Tf
1 TL
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0 0 Td
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5.1071 0 Td
(analysis)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 490.4503 cm
BT
/F9 1 Tf
1 TL
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4.2689 0 Td
(and)Tj
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[()-4()10(")-8()14(#)11()11()]TJ
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(pupae)Tj
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(camera.)Tj
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1.7291 0 Td
(Bownes)Tj
3.3222 0 Td
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0 g
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2.3471 0 Td
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1.6838 0 Td
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1.0148 0 Td
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3.9911 0 Td
(compared)Tj
4.2463 0 Td
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1.0205 0 Td
(the)Tj
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1.474 0 Td
[()-4()10(")-8()14(#)11()11()]TJ
/F9 1 Tf
-33.4826 -1.2982 Td
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3.3732 0 Td
(third)Tj
2.211 0 Td
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(dissected)Tj
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(CCD)Tj
-35.0076 -1.3039 Td
(camera.)Tj
3.3505 0 Td
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1.7801 0 Td
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(two)Tj
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(optic)Tj
2.2337 0 Td
(lobes)Tj
2.262 0 Td
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(and)Tj
1.7348 0 Td
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(\(NIH\))Tj
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-34.4237 -1.2983 Td
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3.7133 0 Td
(wild-type)Tj
3.9912 0 Td
(and)Tj
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1.7291 0 Td
[()-4()10(")-8()14(#)11()11()]TJ
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2.9877 0 Td
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(instar)Tj
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(larval)Tj
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(body)Tj
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(template)Tj
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(1)Tj
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4.473 0 Td
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0.9014 0 Td
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2.5285 0 Td
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2.7042 0 Td
(QPCR)Tj
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(\(Stratagene\))Tj
5.0683 0 Td
(in)Tj
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1.1905 0 Td
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3.4752 0 Td
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3.6567 0 Td
(\(Stratagene\).)Tj
5.2894 0 Td
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1.1622 0 Td
(min)Tj
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