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Tamara J. Abou-Antoun, Javad Nazarian, Anthony Ghanem, Stanislav Vukmanovic, Anthony D. Sandler
Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance
www.plosone.org
www.plosone.org
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2.4435 0 Td
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Q
q
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1 J
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11.9999 0 0 11.9999 200.0125 517.0393 cm
BT
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1 TL
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(proliferation)Tj
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4.1329 0 Td
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2.4491 0 Td
[()]TJ
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(significance)Tj
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3.6567 0 Td
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3.0387 0 Td
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4.337 0 Td
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[()]TJ
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0.7881 0 Td
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1.7291 0 Td
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3.2144 0 Td
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3.6794 0 Td
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Q
q
1 j
1 J
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11.9999 0 0 11.9999 200.0125 367.0299 cm
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1 TL
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2.5323 0 Td
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1 J
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7.4999 0 0 6.6659 446.3433 289.474 cm
BT
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(7)Tj
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1 J
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BT
/F6 1 Tf
1 TL
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ET
Q
q
1 j
1 J
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10 0 0 10 248.9385 259.0299 cm
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1 TL
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BT
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1 TL
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(7)Tj
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Q
q
1 j
1 J
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BT
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(10)Tj
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1 J
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7.4999 0 0 6.6659 312.6047 250.4692 cm
BT
/F6 1 Tf
1 TL
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(6)Tj
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Q
q
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1 TL
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(\(100)Tj
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[()]TJ
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(pAb;1:500)Tj
4.286 0 Td
(Cell)Tj
1.7971 0 Td
(Sig-)Tj
-35.3137 -1.2983 Td
(naling)Tj
2.7269 0 Td
(Technologies;)Tj
5.7599 0 Td
(Cat#)Tj
2.092 0 Td
(9102;)Tj
2.3471 0 Td
(rabbit)Tj
2.6021 0 Td
(pAb;)Tj
2.1544 0 Td
(1:250)Tj
2.347 0 Td
(Santa)Tj
2.4095 0 Td
(Cruz)Tj
2.1996 0 Td
(Technologies;)Tj
5.7543 0 Td
(Cat#)Tj
2.092 0 Td
(sc-101761\))Tj
-32.4848 -1.2983 Td
(\(for)Tj
0.83 0.64 0.02 0 k
1.7234 0 Td
(S2)Tj
1.1735 0 Td
(Fig)Tj
0 g
1.4797 0 Td
(we)Tj
1.3266 0 Td
(used)Tj
2.058 0 Td
(the)Tj
1.474 0 Td
(sampler)Tj
3.3902 0 Td
(kit)Tj
1.2812 0 Td
(rabbit)Tj
2.6079 0 Td
(mAb;)Tj
2.4548 0 Td
(Cell)Tj
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(Signaling)Tj
3.9402 0 Td
(Technologies)Tj
5.5332 0 Td
(Cat#)Tj
2.0862 0 Td
(12651\),)Tj
3.1635 0 Td
(and)Tj
-35.4895 -1.3039 Td
(to)Tj
1.0261 0 Td
(total)Tj
2.0012 0 Td
(and)Tj
1.7292 0 Td
(phosphorylated)Tj
6.4402 0 Td
(expression)Tj
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(of)Tj
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(EGFR)Tj
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(\(Abcam)Tj
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(rabbit)Tj
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(mAb;)Tj
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(1:500)Tj
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(Cat#ab52894;)Tj
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(1:250)Tj
2.347 0 Td
(Cat#ab40815\),)Tj
5.9754 0 Td
(MIA3)Tj
2.6135 0 Td
(\(Abcam)Tj
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(rabbit)Tj
2.6078 0 Td
(pAb;)Tj
2.1543 0 Td
(1:250)Tj
2.3471 0 Td
(Cat#ab97881\),)Tj
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(RhoA)Tj
2.5625 0 Td
(\(Abcam;)Tj
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(rabbit)Tj
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(pAb)Tj
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(1:500)Tj
2.3471 0 Td
(Cat#ab86297;)Tj
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(HeLa)Tj
2.3811 0 Td
(cell)Tj
1.5647 0 Td
(lysates)Tj
2.795 0 Td
(used)Tj
2.0636 0 Td
(as)Tj
1.0148 0 Td
(positive)Tj
3.3108 0 Td
(control\),)Tj
3.6907 0 Td
(cleaved)Tj
3.1521 0 Td
(PARP1)Tj
3.1181 0 Td
(\(1:500;)Tj
2.914 0 Td
(Cell)Tj
-35.9147 -1.3039 Td
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3.9344 0 Td
(Technologies)Tj
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(Cat#)Tj
2.092 0 Td
(5625\),)Tj
2.6872 0 Td
(HDGFR)Tj
3.5886 0 Td
(\(Abcam;)Tj
3.651 0 Td
(rabbit)Tj
2.6079 0 Td
(pAb)Tj
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(1:1000)Tj
2.8233 0 Td
(Cat#210683;)Tj
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(HeLa)Tj
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(control\),)Tj
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(VEGFR2)Tj
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(\(rabbit)Tj
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(pAb;)Tj
2.1543 0 Td
(Abcam)Tj
3.0897 0 Td
(Cat#)Tj
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(ab39256\))Tj
3.8778 0 Td
(survivin)Tj
-32.4168 -1.2983 Td
(\(rabbit)Tj
2.9423 0 Td
(mAb;)Tj
2.4548 0 Td
(1:500,)Tj
2.5738 0 Td
(Cell)Tj
1.8028 0 Td
(Signaling)Tj
3.9345 0 Td
(Technologies)Tj
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(Cat#2808\),)Tj
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(Sox2)Tj
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(\(Abcam;)Tj
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(rabbit)Tj
2.6022 0 Td
(pAb;)Tj
2.1543 0 Td
(1:500;)Tj
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(Cat#137385\))Tj
5.2837 0 Td
(and)Tj
1.7291 0 Td
(CDC2)Tj
2.7609 0 Td
(\(Cell)Tj
2.1374 0 Td
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3.9401 0 Td
(Technology;)Tj
5.159 0 Td
(rabbit)Tj
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(pAb;)Tj
2.1543 0 Td
(1:1000)Tj
2.8233 0 Td
(Cat#)Tj
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(77055\))Tj
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(using)Tj
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(the)Tj
-36.0111 -1.2982 Td
(above)Tj
2.5454 0 Td
(dilutions.)Tj
3.9969 0 Td
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1.7801 0 Td
(blots)Tj
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(were)Tj
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(incubated)Tj
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(with)Tj
1.9956 0 Td
(the)Tj
1.4684 0 Td
(primary)Tj
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(antibody)Tj
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(overnight)Tj
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(at)Tj
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(4C,)Tj
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(washed)Tj
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(5)Tj
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(each)Tj
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(incubated)Tj
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(1)Tj
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(room)Tj
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(anti-rabbit)Tj
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(IgG)Tj
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(HRP-conjugated)Tj
6.9335 0 Td
(\(Cell)Tj
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3.9401 0 Td
(Technology\))Tj
5.2724 0 Td
(antibody)Tj
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(at)Tj
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(a)Tj
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(dilution)Tj
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(of)Tj
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(1:2000.)Tj
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(blots)Tj
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(then)Tj
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(\(4x\))Tj
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(15)Tj
1.1736 0 Td
(mins)Tj
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(each)Tj
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(with)Tj
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(1)Tj
0.6973 0 Td
(x)Tj
0.6803 0 Td
(TBS-Tween)Tj
4.9323 0 Td
(\(0.1%\))Tj
2.8346 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
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(Dura)Tj
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(chemiluminescent)Tj
7.5684 0 Td
(substrate)Tj
3.7984 0 Td
(\(Pierce,)Tj
3.2542 0 Td
(Rockford,)Tj
4.1896 0 Td
(IL\))Tj
1.4399 0 Td
(~2)Tj
1.2756 0 Td
(mins)Tj
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(and)Tj
1.7291 0 Td
(then)Tj
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(exposed.)Tj
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1.3379 0 Td
(control)Tj
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(for)Tj
1.3833 0 Td
(protein)Tj
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(loading)Tj
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(variations,)Tj
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(a)Tj
0.652 0 Td
(primary)Tj
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(antibody)Tj
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(GAPDH)Tj
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(\(Cell)Tj
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(Sig-)Tj
-35.3534 -1.3039 Td
(naling)Tj
2.7269 0 Td
(Technology\))Tj
5.278 0 Td
(was)Tj
1.6951 0 Td
(used,)Tj
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(and)Tj
1.7347 0 Td
(the)Tj
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(total)Tj
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(protein)Tj
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(PDGFRB,)Tj
4.1839 0 Td
(Akt)Tj
1.7008 0 Td
(and)Tj
1.7291 0 Td
(Erk1/2)Tj
2.9367 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
(to)Tj
-36.0394 -1.2983 Td
(measure)Tj
3.5773 0 Td
(the)Tj
1.474 0 Td
(ratio)Tj
2.0976 0 Td
(of)Tj
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(total:phosphorylated)Tj
8.4472 0 Td
(protein,)Tj
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(and)Tj
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(quantitated)Tj
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(by)Tj
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(densitometric)Tj
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(analysis)Tj
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2.3867 0 Td
(ImageLab)Tj
4.1839 0 Td
(5.2)Tj
1.3947 0 Td
(software)Tj
3.5886 0 Td
(Bio-Rad.)Tj
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 418.6771 cm
BT
/F13 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Proteomic)Tj
4.5827 0 Td
(data)Tj
2.0362 0 Td
(and)Tj
1.7906 0 Td
(Statistical)Tj
4.3276 0 Td
(analysis)Tj
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Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 401.7259 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Total)Tj
2.313 0 Td
(protein)Tj
3.1578 0 Td
(was)Tj
1.6951 0 Td
(extracted)Tj
3.8891 0 Td
(from)Tj
2.2053 0 Td
(neuroblastoma)Tj
6.2022 0 Td
(AI)Tj
1.2472 0 Td
(tumorspheres)Tj
5.726 0 Td
(and)Tj
1.7291 0 Td
(AD)Tj
1.6441 0 Td
(adhered)Tj
3.4526 0 Td
(cells,)Tj
2.1486 0 Td
(using)Tj
-35.4101 -1.3039 Td
(RIPPA)Tj
2.982 0 Td
(buffer)Tj
2.6248 0 Td
(\(Sigma-Aldrich,)Tj
6.6444 0 Td
(St.)Tj
1.2189 0 Td
(Louis,)Tj
2.6475 0 Td
(MO\).)Tj
2.4265 0 Td
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2.8913 0 Td
(cell)Tj
1.5591 0 Td
(lysates)Tj
2.7949 0 Td
(\(100ug\))Tj
3.3222 0 Td
(were)Tj
2.1203 0 Td
(reduced)Tj
3.4469 0 Td
(with)Tj
-34.6788 -1.2982 Td
(5mM)Tj
2.4094 0 Td
(DTT)Tj
2.177 0 Td
(in)Tj
1.0318 0 Td
(Laemmli)Tj
3.77 0 Td
(buffer)Tj
2.6249 0 Td
(\(Bio-Rad,)Tj
4.0762 0 Td
(Hercules,)Tj
3.9855 0 Td
(CA\))Tj
1.9105 0 Td
(and)Tj
1.7291 0 Td
(resolved)Tj
3.5377 0 Td
(on)Tj
1.2755 0 Td
(a)Tj
0.652 0 Td
(412%)Tj
2.9197 0 Td
(SDS)Tj
1.9105 0 Td
(poly-)Tj
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(acrylamide)Tj
4.6204 0 Td
(gel.)Tj
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1.7802 0 Td
(gel)Tj
1.3549 0 Td
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1.7008 0 Td
(fixed)Tj
2.1883 0 Td
(in)Tj
1.0375 0 Td
(acetic)Tj
2.4888 0 Td
(acid:methanoal)Tj
6.3382 0 Td
(\(1:1)Tj
1.7348 0 Td
(v/v\))Tj
1.8028 0 Td
(for)Tj
1.3833 0 Td
(30)Tj
1.1736 0 Td
(minutes)Tj
3.4639 0 Td
(and)Tj
1.7291 0 Td
(stained)Tj
-34.3783 -1.3039 Td
(with)Tj
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4.8756 0 Td
(blue)Tj
1.9275 0 Td
(\(1)Tj
1.0318 0 Td
(h\))Tj
1.0885 0 Td
(and)Tj
1.7292 0 Td
(destained)Tj
4.0308 0 Td
(in)Tj
1.0375 0 Td
(water)Tj
2.4321 0 Td
(over)Tj
1.9786 0 Td
(night.)Tj
2.5511 0 Td
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2.1713 0 Td
(protein)Tj
3.1578 0 Td
(lane)Tj
1.8709 0 Td
(was)Tj
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(then)Tj
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(cut)Tj
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(into)Tj
1.8425 0 Td
(36)Tj
1.1678 0 Td
(bands)Tj
2.5965 0 Td
(and)Tj
1.7348 0 Td
(each)Tj
2.024 0 Td
(band)Tj
2.2393 0 Td
(was)Tj
1.7008 0 Td
(subjected)Tj
3.9685 0 Td
(to)Tj
1.0261 0 Td
(in-gel)Tj
2.5172 0 Td
(digestion)Tj
3.8891 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0478 0 Td
(before)Tj
2.7439 0 Td
([)Tj
0.83 0.64 0.02 0 k
(6)Tj
0 g
(].)Tj
1.5988 0 Td
(Briefly,)Tj
-34.1119 -1.2982 Td
(proteins)Tj
3.5035 0 Td
(from)Tj
2.1941 0 Td
(each)Tj
2.0239 0 Td
(band)Tj
2.228 0 Td
(were)Tj
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(digested)Tj
3.5036 0 Td
(with)Tj
1.9899 0 Td
(trypsin)Tj
3.0273 0 Td
(and)Tj
1.7235 0 Td
(sequenced)Tj
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(by)Tj
1.1735 0 Td
(Liquid)Tj
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-30.6933 -1.304 Td
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2.2677 0 Td
(Spectrometry/)Tj
5.9357 0 Td
(Mass)Tj
2.2677 0 Td
(Spectrometry)Tj
5.6012 0 Td
(\(LC-MS/MS\))Tj
5.5218 0 Td
(and)Tj
1.7292 0 Td
(identified)Tj
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(by)Tj
1.1792 0 Td
(searching)Tj
4.0421 0 Td
(against)Tj
-32.6038 -1.2982 Td
(murine)Tj
3.1747 0 Td
(databases.)Tj
4.2293 0 Td
(We)Tj
1.6101 0 Td
(used)Tj
2.0579 0 Td
(ProteoIQ)Tj
3.9742 0 Td
(software)Tj
3.5829 0 Td
(\(Bioinquire,)Tj
5.0513 0 Td
(Atlanta)Tj
3.1635 0 Td
(GA\),)Tj
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(our)Tj
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(analysis)Tj
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(previously)Tj
4.3483 0 Td
(described)Tj
4.0535 0 Td
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(7)Tj
0 g
(,)Tj
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(8)Tj
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(].)Tj
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(advantage)Tj
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(is)Tj
0.8447 0 Td
(its)Tj
1.1509 0 Td
(ability)Tj
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(to)Tj
1.0261 0 Td
(extract)Tj
2.931 0 Td
(the)Tj
1.474 0 Td
(spectral)Tj
-32.0539 -1.3039 Td
(counts)Tj
2.8856 0 Td
(\(SCs\))Tj
2.3981 0 Td
(for)Tj
1.389 0 Td
(each)Tj
2.0296 0 Td
(peptide)Tj
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(and)Tj
1.7292 0 Td
(compare)Tj
3.719 0 Td
(it)Tj
0.7824 0 Td
(across)Tj
2.6815 0 Td
(several)Tj
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(samples.)Tj
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(initial)Tj
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(analysis)Tj
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(showed)Tj
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(a)Tj
0.6577 0 Td
(linear)Tj
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(expression)Tj
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(of)Tj
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(the)Tj
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(majority)Tj
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(of)Tj
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(proteins.)Tj
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(total)Tj
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(of)Tj
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(1,069)Tj
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(proteins)Tj
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(were)Tj
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(identified)Tj
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(the)Tj
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(following)Tj
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(stringent)Tj
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(criteria:)Tj
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(quantification)Tj
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(performed)Tj
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(using)Tj
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(software)Tj
3.5829 0 Td
(\(Bioinquire\))Tj
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(spectral)Tj
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(count)Tj
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(data.)Tj
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1.7801 0 Td
(following)Tj
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(stringent)Tj
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(filtration)Tj
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(criteria)Tj
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(2)Tj
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(more)Tj
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(peptides/protein,)Tj
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(10)Tj
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(or)Tj
1.0998 0 Td
(more)Tj
2.3414 0 Td
(spectra/protein,)Tj
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(minimum)Tj
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(peptide)Tj
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(probability-)Tj
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(based)Tj
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(score)Tj
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[()]TJ
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0 Tc
0.771 0 Td
()Tj
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-0.004 Tc
0.9921 0 Td
(.05,)Tj
1.6215 0 Td
(and)Tj
1.7291 0 Td
(a)Tj
0.6519 0 Td
(variable)Tj
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(threshold)Tj
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1.0148 0 Td
[()25()7()11()-9()]TJ
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2.4094 0 Td
(versus)Tj
2.7212 0 Td
(charge)Tj
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(state)Tj
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(\()Tj
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[()25()7()11()-9()]TJ
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2.7496 0 Td
(=)Tj
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