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Antonio Castro-Castro, Olivia Muriel, Miguel A. del Pozo, Xosé R. Bustelo
Characterization of Novel Molecular Mechanisms Favoring Rac1 Membrane Translocation
www.plosone.org
www.plosone.org
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(polyclonal)Tj
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(antibodies)Tj
4.3596 0 Td
(to)Tj
1.0261 0 Td
(Rho)Tj
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(GDI)Tj
2.0126 0 Td
(\(clones)Tj
3.067 0 Td
(K-21)Tj
2.194 0 Td
(and)Tj
1.7348 0 Td
(A-20\),)Tj
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(GADPH,)Tj
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(and)Tj
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(GST)Tj
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(were)Tj
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(Santa)Tj
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(Cruz)Tj
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(polyclonal)Tj
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(antibodies)Tj
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(phospho-Erk)Tj
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(and)Tj
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(EGFP)Tj
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(Cell)Tj
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1.7177 0 Td
(Clontech,)Tj
4.0762 0 Td
(respectively.)Tj
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(polyclonal)Tj
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(antibodies)Tj
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(CD98)Tj
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(phalloidin,)Tj
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Q
q
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1 J
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11.9999 0 0 11.9999 200.0125 529.0582 cm
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1 TL
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Q
q
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1 J
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1 J
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7.4999 0 0 6.6659 478.148 516.5291 cm
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1 TL
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Q
q
1 j
1 J
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q
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7.4999 0 0 6.6659 360.5102 503.4897 cm
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1 TL
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Q
q
1 j
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Q
q
1 j
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Q
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1 J
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/F7 1 Tf
1 TL
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(K44A)Tj
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Q
q
1 j
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(TCC)Tj
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(ATG)Tj
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(Nagoya)Tj
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(University)Tj
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(generate)Tj
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(kit,)Tj
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(Agilent)Tj
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(Technologies\))Tj
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(to)Tj
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(generate)Tj
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(the)Tj
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Q
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1 j
1 J
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7.4999 0 0 6.6659 282.444 178.4692 cm
BT
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1 TL
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Q
q
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1 J
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1 TL
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(\(ii\))Tj
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(the)Tj
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[( )11(\n\n\r)]TJ
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Q
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(incubated)Tj
4.1726 0 Td
(for)Tj
1.389 0 Td
(30)Tj
-36.4363 -1.2983 Td
(min)Tj
1.8538 0 Td
(at)Tj
0.9524 0 Td
(room)Tj
2.4265 0 Td
(temperature,)Tj
5.3518 0 Td
(and)Tj
1.7291 0 Td
(added)Tj
2.6645 0 Td
(onto)Tj
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(cells.)Tj
2.143 0 Td
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2.2847 0 Td
(culturing)Tj
3.8948 0 Td
(for)Tj
1.389 0 Td
(2436)Tj
2.6418 0 Td
(hours,)Tj
2.7383 0 Td
(cells)Tj
1.9219 0 Td
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3.1747 0 Td
(with)Tj
2.0013 0 Td
(chilled)Tj
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(phosphate)Tj
4.3313 0 Td
(buffered)Tj
3.5773 0 Td
(saline)Tj
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(solution,)Tj
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(1)Tj
0.6973 0 Td
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(hypo-)Tj
-35.1777 -1.3039 Td
(tonic)Tj
2.262 0 Td
(solution)Tj
3.4866 0 Td
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(\(20)Tj
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(mM)Tj
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(Tris-HCl)Tj
3.8607 0 Td
([pH)Tj
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(7.5],)Tj
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(10)Tj
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(10)Tj
1.1735 0 Td
(mM)Tj
1.9332 0 Td
(NaCl,)Tj
2.5229 0 Td
(5)Tj
0.6973 0 Td
(mM)Tj
1.9332 0 Td
(MgCl)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 551.3952 569.1401 cm
BT
/F7 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 554.9102 570.7842 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4478 0 Td
(1)Tj
-35.9373 -1.2982 Td
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1.9332 0 Td
(DTT,)Tj
2.4038 0 Td
(1)Tj
0.6916 0 Td
(mM)Tj
1.9333 0 Td
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ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 281.3669 556.1574 cm
BT
/F7 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 284.8818 557.8015 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(VO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 299.3385 556.1574 cm
BT
/F7 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 302.8535 557.8015 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4478 0 Td
(10)Tj
1.1679 0 Td
(mM)Tj
/F11 1 Tf
1.9332 0 Td
()Tj
/F7 1 Tf
(-glycerophosphate)Tj
8.0617 0 Td
(and)Tj
1.7348 0 Td
(the)Tj
1.4683 0 Td
(C)Tj
/F13 1 Tf
[()]TJ
/F7 1 Tf
(mplete)Tj
4.1783 0 Td
(protease)Tj
3.5602 0 Td
(inhibitor)Tj
-32.8362 -1.2982 Td
(mix)Tj
1.7688 0 Td
([Roche)Tj
3.0671 0 Td
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(Sciences]\),)Tj
4.4729 0 Td
(and)Tj
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(homogenized)Tj
5.6522 0 Td
(by)Tj
1.1792 0 Td
(passing)Tj
3.1748 0 Td
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0.9524 0 Td
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1.9843 0 Td
(15)Tj
1.1678 0 Td
(times)Tj
2.3925 0 Td
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3.4412 0 Td
(a)Tj
0.6576 0 Td
(blunt,)Tj
-33.3748 -1.3039 Td
(20-gauge)Tj
3.8381 0 Td
(needle)Tj
2.812 0 Td
(fitted)Tj
2.3244 0 Td
(to)Tj
1.0204 0 Td
(a)Tj
0.6576 0 Td
(2)Tj
0.6916 0 Td
(ml)Tj
1.2869 0 Td
(syringe.)Tj
3.3392 0 Td
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3.0898 0 Td
(were)Tj
2.1203 0 Td
(then)Tj
2.0182 0 Td
(kept)Tj
1.9559 0 Td
(on)Tj
1.2756 0 Td
(ice)Tj
1.3379 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1736 0 Td
(min,)Tj
2.0806 0 Td
(centrifuged)Tj
-32.4054 -1.2983 Td
(at)Tj
0.9525 0 Td
(14,000)Tj
2.8232 0 Td
(rpm)Tj
1.9276 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1735 0 Td
(min)Tj
1.8539 0 Td
(at)Tj
0.9523 0 Td
(4C)Tj
1.7518 0 Td
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1.0261 0 Td
(remove)Tj
3.2202 0 Td
(unbroken)Tj
4.1499 0 Td
(cells)Tj
1.9219 0 Td
(and)Tj
1.7291 0 Td
(nuclei.)Tj
2.88 0 Td
(The)Tj
1.7858 0 Td
(supernatants)Tj
-29.5311 -1.2983 Td
(obtained)Tj
3.7304 0 Td
(in)Tj
1.0375 0 Td
(that)Tj
1.7801 0 Td
(step)Tj
1.8198 0 Td
(were)Tj
2.1202 0 Td
(transferred)Tj
4.6375 0 Td
(into)Tj
1.8425 0 Td
(polycarbonate)Tj
5.8904 0 Td
(tubes)Tj
2.3414 0 Td
(\(Beckman\))Tj
4.6261 0 Td
(and)Tj
1.7291 0 Td
(subjected)Tj
3.9685 0 Td
(to)Tj
-35.5235 -1.3039 Td
(centrifugation)Tj
5.8847 0 Td
(at)Tj
0.9524 0 Td
(100,000)Tj
3.2995 0 Td
(xg)Tj
1.1508 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6916 0 Td
(hour)Tj
2.1543 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(refrigerated)Tj
4.8869 0 Td
(ultracentrifuge)Tj
6.1341 0 Td
(\(Beckman\).)Tj
4.8529 0 Td
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2.2847 0 Td
(the)Tj
-35.3704 -1.2982 Td
(centrifugation,)Tj
6.1115 0 Td
(the)Tj
1.4683 0 Td
(cytosol-containing)Tj
7.6761 0 Td
(supernatant)Tj
4.9833 0 Td
(was)Tj
1.7007 0 Td
(removed)Tj
3.7474 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
1.4684 0 Td
(crude)Tj
2.4945 0 Td
(membrane)Tj
4.5694 0 Td
(pel-)Tj
-35.9487 -1.2983 Td
(let)Tj
1.1792 0 Td
(gently)Tj
2.6476 0 Td
(washed)Tj
3.1691 0 Td
(with)Tj
1.9899 0 Td
(hypotonic)Tj
4.2632 0 Td
(lysis)Tj
1.9048 0 Td
(buffer)Tj
2.6136 0 Td
(and)Tj
1.7234 0 Td
(recollected)Tj
4.5241 0 Td
(by)Tj
1.1792 0 Td
(ultracentrifugation.)Tj
7.9823 0 Td
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-33.1764 -1.2983 Td
(fractions)Tj
3.7191 0 Td
(were)Tj
2.1203 0 Td
(then)Tj
2.0182 0 Td
(assayed)Tj
3.2144 0 Td
(for)Tj
1.3889 0 Td
(total)Tj
2.0013 0 Td
(protein)Tj
3.1521 0 Td
(content)Tj
3.2712 0 Td
(using)Tj
2.3867 0 Td
(the)Tj
1.474 0 Td
(Bradford)Tj
3.8268 0 Td
(method)Tj
3.3222 0 Td
(\(Bio-Rad\))Tj
4.1895 0 Td
(and)Tj
-36.0847 -1.3039 Td
(subjected)Tj
3.9685 0 Td
(to)Tj
1.0261 0 Td
(immunoblotting.)Tj
7.0695 0 Td
(As)Tj
1.2642 0 Td
(control,)Tj
3.3506 0 Td
(we)Tj
1.3266 0 Td
(analyzed)Tj
3.702 0 Td
(by)Tj
1.1792 0 Td
(aliquots)Tj
3.3675 0 Td
(of)Tj
1.0148 0 Td
(cell)Tj
1.5648 0 Td
(lysates)Tj
2.7949 0 Td
(taken)Tj
2.4208 0 Td
(before)Tj
-34.0495 -1.2983 Td
(the)Tj
1.4684 0 Td
(ultracentrifugation)Tj
7.7668 0 Td
(step)Tj
1.8198 0 Td
(as)Tj
1.0148 0 Td
(above.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 399.1748 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Immunoblotting)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 382.2236 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Protein)Tj
3.1917 0 Td
(samples)Tj
3.3846 0 Td
(obtained)Tj
3.7304 0 Td
(as)Tj
1.0148 0 Td
(indicated)Tj
3.9401 0 Td
(in)Tj
1.0375 0 Td
(each)Tj
2.0239 0 Td
(experimental)Tj
5.4595 0 Td
(procedure)Tj
4.3143 0 Td
(were)Tj
2.1203 0 Td
(denatured)Tj
4.3029 0 Td
(by)Tj
1.1849 0 Td
(boil-)Tj
-35.7049 -1.3039 Td
(ing)Tj
1.5023 0 Td
(in)Tj
1.0318 0 Td
(SDS-PAGE)Tj
4.7849 0 Td
(sample)Tj
3.0217 0 Td
(buffer,)Tj
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(separated)Tj
4.0138 0 Td
(electrophoretically,)Tj
7.852 0 Td
(and)Tj
1.7348 0 Td
(transferred)Tj
4.6374 0 Td
(onto)Tj
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(nitrocel-)Tj
-33.5109 -1.2982 Td
(lulose)Tj
2.5284 0 Td
(filters)Tj
2.4775 0 Td
(using)Tj
2.3868 0 Td
(the)Tj
1.474 0 Td
(iBlot)Tj
2.1146 0 Td
(Dry)Tj
1.7858 0 Td
(Blotting)Tj
3.4299 0 Td
(System)Tj
3.0501 0 Td
(\(Thermo)Tj
3.8211 0 Td
(Fisher)Tj
2.6985 0 Td
(Scientific\).)Tj
4.4674 0 Td
(Membranes)Tj
5.0003 0 Td
(were)Tj
-35.2344 -1.2983 Td
(blocked)Tj
3.3505 0 Td
(in)Tj
1.0375 0 Td
(either)Tj
2.5284 0 Td
(5%)Tj
1.4514 0 Td
(non-fat)Tj
3.1974 0 Td
(dry)Tj
1.5761 0 Td
(milk)Tj
2.0466 0 Td
(\(when)Tj
2.7439 0 Td
(immunoblotted)Tj
6.5196 0 Td
(with)Tj
1.9956 0 Td
(standard)Tj
3.7247 0 Td
(antibodies\))Tj
4.6998 0 Td
(or)Tj
1.0999 0 Td
(5%)Tj
-35.9714 -1.3039 Td
(bovine)Tj
2.9253 0 Td
(serum)Tj
2.7269 0 Td
(albumin)Tj
3.566 0 Td
(\(Sigma,)Tj
3.2484 0 Td
(when)Tj
2.4038 0 Td
(using)Tj
2.3924 0 Td
(phospho-specific)Tj
7.0016 0 Td
(antibodies\))Tj
4.7054 0 Td
(in)Tj
1.0319 0 Td
(25)Tj
1.1735 0 Td
(mM)Tj
1.9332 0 Td
(Tris-HCl)Tj
-33.1084 -1.2983 Td
(\(pH)Tj
1.8425 0 Td
(8.0\),)Tj
1.9615 0 Td
(150)Tj
1.6498 0 Td
(mM)Tj
1.9332 0 Td
(NaCl,)Tj
2.5228 0 Td
(0.1%)Tj
2.1543 0 Td
(Tween-20)Tj
4.2123 0 Td
(\(TBS-T\))Tj
3.5263 0 Td
(for)Tj
1.3889 0 Td
(1)Tj
0.6973 0 Td
(hour)Tj
2.1544 0 Td
(and)Tj
1.7291 0 Td
(then)Tj
2.0182 0 Td
(incubated)Tj
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(overnight)Tj
4.0818 0 Td
(at)Tj
-36.0507 -1.2982 Td
(4C)Tj
1.7517 0 Td
(with)Tj
1.9956 0 Td
(the)Tj
1.4684 0 Td
(appropriate)Tj
4.8812 0 Td
(primary)Tj
3.4526 0 Td
(antibody)Tj
3.7644 0 Td
(diluted)Tj
3.0387 0 Td
(in)Tj
1.0375 0 Td
(blocking)Tj
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(buffer.)Tj
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2.2847 0 Td
(three)Tj
2.2621 0 Td
(washes)Tj
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(TBS-T,)Tj
3.0727 0 Td
(membranes)Tj
4.9266 0 Td
(were)Tj
2.1259 0 Td
(incubated)Tj
4.1726 0 Td
(with)Tj
2.0013 0 Td
(horseradish)Tj
4.8982 0 Td
(peroxidase-conjugated)Tj
9.2806 0 Td
(to)Tj
1.0261 0 Td
(either)Tj
2.5285 0 Td
(protein)Tj
-34.0325 -1.2982 Td
(A)Tj
0.9014 0 Td
(or)Tj
1.0998 0 Td
(the)Tj
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4.8813 0 Td
(secondary)Tj
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(antibody)Tj
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(\(1:5,000)Tj
3.3902 0 Td
(dilution\))Tj
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(for)Tj
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(1)Tj
0.6916 0 Td
(hour)Tj
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(at)Tj
0.9525 0 Td
(room)Tj
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(temperature.)Tj
-31.1355 -1.2983 Td
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7.0128 0 Td
(bands)Tj
2.5965 0 Td
(were)Tj
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(developed)Tj
4.269 0 Td
(using)Tj
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(a)Tj
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(standard)Tj
3.7247 0 Td
(chemoluminescent)Tj
7.8066 0 Td
(method)Tj
3.3222 0 Td
(\(ECL,)Tj
-33.9021 -1.2983 Td
(GE)Tj
1.5023 0 Td
(Healthcare)Tj
4.5694 0 Td
(Life)Tj
1.7405 0 Td
(Biosciences\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 223.5968 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(siRNA)Tj
3.0236 0 Td
(knockdown)Tj
5.1638 0 Td
(experiments)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 206.5889 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Transcript)Tj
4.422 0 Td
(knockdowns)Tj
5.3234 0 Td
(were)Tj
2.1203 0 Td
(done)Tj
2.228 0 Td
(by)Tj
1.1792 0 Td
(transfecting)Tj
4.9493 0 Td
(siRNAs)Tj
3.2485 0 Td
(\(200)Tj
1.9899 0 Td
(pmol\))Tj
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(targeting)Tj
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[()13()]TJ
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(\(M-032006-01-0005,)Tj
8.4358 0 Td
(On-TargetPlus)Tj
6.1625 0 Td
(collection,)Tj
4.337 0 Td
(GE)Tj
1.5023 0 Td
(Dharmacon\))Tj
5.3631 0 Td
([)Tj
0.83 0.64 0.02 0 k
(40)Tj
0 g
(],)Tj
/F12 1 Tf
2.0806 0 Td
[()-6()13()8(\r)]TJ
/F7 1 Tf
3.9345 0 Td
(\(L-012771-00-)Tj
-31.8158 -1.2983 Td
(0005,)Tj
2.347 0 Td
(On-TargetPlus)Tj
6.1625 0 Td
(SmartPool)Tj
4.439 0 Td
(collection,)Tj
4.337 0 Td
(Thermo)Tj
3.4809 0 Td
(Fisher)Tj
2.6986 0 Td
(Scientific\))Tj
4.2406 0 Td
(or)Tj
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1.0998 0 Td
[()]TJ
/F7 1 Tf
2.4662 0 Td
(\(L-003542-00-)Tj
-31.2716 -1.3039 Td
(0005,)Tj
2.347 0 Td
(On-TargetPlus)Tj
6.1625 0 Td
(SmartPools)Tj
4.8019 0 Td
(collection,)Tj
4.3313 0 Td
(Thermo)Tj
3.4809 0 Td
(Fisher)Tj
2.6986 0 Td
(Scientific\))Tj
4.2462 0 Td
(mRNAs.)Tj
3.668 0 Td
(As)Tj
1.2643 0 Td
(control,)Tj
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(we)Tj
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(used)Tj
2.0579 0 Td
(a)Tj
0.652 0 Td
(scrambled)Tj
4.3369 0 Td
(siRNA)Tj
2.8914 0 Td
(\(D-001810-10-05,)Tj
7.3303 0 Td
(On-TargetPlus)Tj
6.1625 0 Td
(Non-targeting)Tj
5.93 0 Td
(pool,)Tj
2.2224 0 Td
(GE)Tj
1.508 0 Td
(Dharma-)Tj
-33.0914 -1.2982 Td
(con\).)Tj
2.262 0 Td
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5.6636 0 Td
(were)Tj
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(made)Tj
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(using)Tj
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(Lipofectamine)Tj
5.9754 0 Td
(2000)Tj
2.1203 0 Td
(as)Tj
1.0148 0 Td
(above.)Tj
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Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 112.9889 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Co-immunoprecipitat)Tj
9.1134 0 Td
(ion)Tj
1.5355 0 Td
(experiments)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 95.9811 cm
BT
/F7 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Lipofectamine)Tj
5.9753 0 Td
(2000-trasfected)Tj
6.3213 0 Td
(COS1)Tj
2.5795 0 Td
(cells)Tj
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(cultured)Tj
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(2436)Tj
2.6419 0 Td
(hours,)Tj
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(washed)Tj
3.1748 0 Td
(with)Tj
2.0012 0 Td
(chilled)Tj
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(phosphate)Tj
4.3313 0 Td
(buffered)Tj
3.5773 0 Td
(saline)Tj
2.5001 0 Td
(solution,)Tj
3.7077 0 Td
(disrupted)Tj
4.0422 0 Td
(in)Tj
1.0374 0 Td
(1)Tj
0.6974 0 Td
(ml)Tj
1.2812 0 Td
(of)Tj
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(20)Tj
1.1736 0 Td
(mM)Tj
1.9332 0 Td
(Tris-HCl)Tj
3.8551 0 Td
(\(pH)Tj
1.8481 0 Td
(7.5\),)Tj
1.9616 0 Td
(150)Tj
1.6441 0 Td
(mM)Tj
ET
Q
q
1 j
1 J
0 w
576 737.1 m
36 737.1 l
36 737.6 l
576 737.6 l
f*
36 741.2598 107.1496 23.6976 re
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