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Barbara Lipert, Mateusz Wilamowski, Andrzej Gorecki, Jolanta Jura
MCPIP1, alias Regnase-1 binds and cleaves mRNA of C/EBPβ
www.plosone.org
www.plosone.org
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2.7326 0 Td
(pFLAG-CMV-2)Tj
6.6273 0 Td
(vector)Tj
2.6986 0 Td
(\(E7033;)Tj
3.2598 0 Td
(SIGMA\).)Tj
3.8948 0 Td
(To)Tj
1.3379 0 Td
(per-)Tj
-35.4441 -1.2983 Td
(form)Tj
2.2053 0 Td
(crosslinking,)Tj
5.3064 0 Td
(cells)Tj
1.9219 0 Td
(on)Tj
1.2756 0 Td
(the)Tj
1.4683 0 Td
(plates)Tj
2.5002 0 Td
(were)Tj
2.1259 0 Td
(treated)Tj
2.9934 0 Td
(with)Tj
1.9956 0 Td
(formaldehyde)Tj
5.7713 0 Td
(\(432173111;)Tj
5.0683 0 Td
(POCh,)Tj
-32.6322 -1.2983 Td
(Poland\))Tj
3.3788 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(final)Tj
2.0069 0 Td
(concentration)Tj
5.8166 0 Td
(of)Tj
1.0148 0 Td
(1%)Tj
1.457 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1736 0 Td
(minutes.)Tj
3.6907 0 Td
(Cells)Tj
2.1599 0 Td
(were)Tj
2.1203 0 Td
(collected)Tj
3.7417 0 Td
(from)Tj
2.2054 0 Td
(plates)Tj
2.5001 0 Td
(and)Tj
-34.3386 -1.3039 Td
(washed)Tj
3.1747 0 Td
(twice)Tj
2.3188 0 Td
(using)Tj
2.3867 0 Td
(cold)Tj
1.9276 0 Td
(PBS)Tj
1.8311 0 Td
(buffer)Tj
2.6306 0 Td
(with)Tj
1.9955 0 Td
(the)Tj
1.4684 0 Td
(addition)Tj
3.6 0 Td
(of)Tj
1.0148 0 Td
(a)Tj
0.6519 0 Td
(protease)Tj
3.5546 0 Td
(inhibitors)Tj
4.1443 0 Td
(cocktail)Tj
3.3165 0 Td
(\(P8340,)Tj
-34.0155 -1.2982 Td
(Sigma-Aldrich\).)Tj
6.6443 0 Td
(Cells)Tj
2.16 0 Td
(were)Tj
2.1203 0 Td
(centrifuged)Tj
4.7849 0 Td
(at)Tj
0.9524 0 Td
(120)Tj
1.6497 0 Td
(RCF)Tj
2.0296 0 Td
(for)Tj
1.389 0 Td
(10)Tj
1.1679 0 Td
(minutes)Tj
3.4696 0 Td
(at)Tj
0.9524 0 Td
(room)Tj
2.4208 0 Td
(temperature)Tj
5.1306 0 Td
(and)Tj
-34.8715 -1.2983 Td
(lysed)Tj
2.2336 0 Td
(in)Tj
1.0318 0 Td
(a)Tj
0.6577 0 Td
(buffer)Tj
2.6248 0 Td
(containing)Tj
4.5241 0 Td
(50)Tj
1.1735 0 Td
(mM)Tj
1.9333 0 Td
(HEPES,)Tj
3.3845 0 Td
(400)Tj
1.6498 0 Td
(mM)Tj
1.9332 0 Td
(NaCl,)Tj
2.5228 0 Td
(1)Tj
0.6973 0 Td
(mM)Tj
1.9332 0 Td
(EDTA,)Tj
3.0444 0 Td
(1)Tj
0.6973 0 Td
(mM)Tj
1.9276 0 Td
(DTT,)Tj
2.4094 0 Td
(10%)Tj
-34.3783 -1.3039 Td
(glycerol,)Tj
3.5829 0 Td
(0.5%)Tj
2.1543 0 Td
(Triton)Tj
2.8233 0 Td
(X-100)Tj
2.6476 0 Td
(\(pH)Tj
1.8481 0 Td
(7.5\),)Tj
1.9616 0 Td
(supplemented)Tj
5.8507 0 Td
(with)Tj
2.0012 0 Td
(an)Tj
1.2019 0 Td
(RNase)Tj
2.795 0 Td
(inhibitor)Tj
3.7813 0 Td
(\(0371000;)Tj
4.6318 0 Td
(A&A)Tj
-35.2797 -1.2983 Td
(Biotechnology\))Tj
6.3155 0 Td
(and)Tj
1.7291 0 Td
(protease)Tj
3.5546 0 Td
(inhibitor)Tj
3.7814 0 Td
(cocktail)Tj
3.3166 0 Td
(\(P8340,)Tj
3.2484 0 Td
(Sigma-Aldrich\).)Tj
6.6444 0 Td
(The)Tj
1.7802 0 Td
(pellet)Tj
2.3754 0 Td
(was)Tj
1.7007 0 Td
(re-sus-)Tj
-34.4463 -1.2983 Td
(pended)Tj
3.1861 0 Td
(in)Tj
1.0374 0 Td
(the)Tj
1.4684 0 Td
(lysis)Tj
1.9105 0 Td
(buffer,)Tj
2.8517 0 Td
(frozen)Tj
2.7722 0 Td
(in)Tj
1.0318 0 Td
(liquid)Tj
2.5625 0 Td
(nitrogen)Tj
3.651 0 Td
(and)Tj
1.7292 0 Td
(then)Tj
2.0182 0 Td
(thawed)Tj
3.1181 0 Td
(rapidly)Tj
3.0387 0 Td
(to)Tj
1.0262 0 Td
(promote)Tj
3.6623 0 Td
(cell)Tj
-35.0643 -1.3039 Td
(disruption.)Tj
4.6374 0 Td
(Subsequent)Tj
4.8189 0 Td
(immunoprecipitation)Tj
8.8497 0 Td
(of)Tj
1.0148 0 Td
(MCPIP1)Tj
3.7133 0 Td
(complexes)Tj
4.405 0 Td
(with)Tj
1.9956 0 Td
(RNA)Tj
2.2621 0 Td
(was)Tj
1.7007 0 Td
(done)Tj
2.2281 0 Td
(in)Tj
1.0318 0 Td
(IP)Tj
-36.6574 -1.2982 Td
(buffer:)Tj
2.8516 0 Td
(50)Tj
1.1679 0 Td
(mM)Tj
1.9332 0 Td
(HEPES,)Tj
3.3845 0 Td
(150)Tj
1.6498 0 Td
(mM)Tj
1.9332 0 Td
(NaCl,)Tj
2.5228 0 Td
(1)Tj
0.6973 0 Td
(mM)Tj
1.9332 0 Td
(EDTA,)Tj
3.0444 0 Td
(2)Tj
0.6973 0 Td
(mM)Tj
1.9333 0 Td
(DTT,)Tj
2.4037 0 Td
(10%)Tj
1.9276 0 Td
(glycerol,)Tj
3.5829 0 Td
(0.5%)Tj
2.1544 0 Td
(Triton)Tj
-33.8171 -1.2983 Td
(X-100)Tj
2.6475 0 Td
(\(pH)Tj
1.8482 0 Td
(7.5\))Tj
1.7348 0 Td
(supplemented)Tj
5.8563 0 Td
(with)Tj
1.9956 0 Td
(an)Tj
1.2019 0 Td
(RNase)Tj
2.7949 0 Td
(inhibitor)Tj
3.7814 0 Td
(and)Tj
1.7291 0 Td
(protease)Tj
3.5603 0 Td
(inhibitor)Tj
3.7814 0 Td
(cocktail.)Tj
3.5376 0 Td
(Sam-)Tj
-34.469 -1.3039 Td
(ples)Tj
1.7688 0 Td
(were)Tj
2.1203 0 Td
(incubated)Tj
4.1782 0 Td
(overnight)Tj
4.0819 0 Td
(with)Tj
1.9955 0 Td
(50)Tj
/F17 1 Tf
1.1736 0 Td
[()]TJ
/F9 1 Tf
(l)Tj
0.9751 0 Td
(Protein)Tj
3.1918 0 Td
(A)Tj
0.9071 0 Td
(agarose)Tj
3.2144 0 Td
(\(11719408001;)Tj
6.0151 0 Td
(Roche\))Tj
3.0671 0 Td
(and)Tj
1.7291 0 Td
(30)Tj
/F17 1 Tf
1.1679 0 Td
[()]TJ
/F9 1 Tf
(g)Tj
-35.5859 -1.2983 Td
(monoclonal)Tj
5.0059 0 Td
(anti-FLAG)Tj
4.5864 0 Td
(M2)Tj
1.5874 0 Td
(antibody)Tj
3.7644 0 Td
(1:1000)Tj
2.8233 0 Td
(\(F1804;)Tj
3.2145 0 Td
(SIGMA\).)Tj
3.8947 0 Td
(Next,)Tj
2.3698 0 Td
(samples)Tj
3.3789 0 Td
(were)Tj
2.1259 0 Td
(washed)Tj
3.1748 0 Td
(five)Tj
-35.926 -1.2982 Td
(times)Tj
2.3867 0 Td
(in)Tj
1.0375 0 Td
(1)Tj
0.6973 0 Td
(ml)Tj
1.2813 0 Td
(IP)Tj
1.1225 0 Td
(buffer,)Tj
2.8516 0 Td
(followed)Tj
3.6453 0 Td
(by)Tj
1.1849 0 Td
(centrifugation)Tj
5.8904 0 Td
(at)Tj
0.9467 0 Td
(300)Tj
1.6498 0 Td
(RCF)Tj
2.0352 0 Td
(for)Tj
1.3833 0 Td
(2)Tj
0.6974 0 Td
(minutes.)Tj
3.6906 0 Td
(For)Tj
1.6271 0 Td
(proteinase)Tj
4.371 0 Td
(K)Tj
-36.4986 -1.2983 Td
(treatment)Tj
4.1328 0 Td
(\(V302B,)Tj
3.4866 0 Td
(Promega\),)Tj
4.3654 0 Td
(50)Tj
/F17 1 Tf
1.1735 0 Td
[()]TJ
/F9 1 Tf
(g)Tj
1.1905 0 Td
(of)Tj
1.0148 0 Td
(enzyme)Tj
3.3052 0 Td
(was)Tj
1.6951 0 Td
(added)Tj
2.6646 0 Td
(to)Tj
1.0261 0 Td
(samples.)Tj
3.6056 0 Td
(Reverse)Tj
3.2996 0 Td
(of)Tj
1.0148 0 Td
(cross-linking)Tj
-31.9746 -1.3039 Td
(was)Tj
1.6951 0 Td
(done)Tj
2.228 0 Td
(in)Tj
1.0374 0 Td
(RIP)Tj
1.7405 0 Td
(buffer:)Tj
2.8516 0 Td
(50)Tj
1.1679 0 Td
(mM)Tj
1.9332 0 Td
(HEPES,)Tj
3.3902 0 Td
(100)Tj
1.6441 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
(EDTA,)Tj
3.0444 0 Td
(10)Tj
1.1735 0 Td
(mM)Tj
1.9332 0 Td
(DTT,)Tj
2.4038 0 Td
(10%)Tj
1.9276 0 Td
(glyc-)Tj
-35.2571 -1.2983 Td
(erol,)Tj
1.9899 0 Td
(1,5%)Tj
2.1543 0 Td
(SDS,)Tj
2.1373 0 Td
(0.5%)Tj
2.1486 0 Td
(Triton)Tj
2.8233 0 Td
(X-100)Tj
2.6532 0 Td
(\(pH)Tj
1.8425 0 Td
(7.5\))Tj
1.7405 0 Td
(supplemented)Tj
5.8507 0 Td
(with)Tj
2.0012 0 Td
(RNase)Tj
2.795 0 Td
(inhibitor.)Tj
4.0081 0 Td
(Samples)Tj
3.4923 0 Td
(were)Tj
-35.6369 -1.2983 Td
(incubated)Tj
4.1782 0 Td
(in)Tj
1.0318 0 Td
(RIP)Tj
1.7405 0 Td
(buffer)Tj
2.6248 0 Td
(at)Tj
0.9525 0 Td
(70C)Tj
2.228 0 Td
(for)Tj
1.3889 0 Td
(1)Tj
0.6917 0 Td
(hour,)Tj
2.3811 0 Td
(then)Tj
2.0182 0 Td
(supernatants)Tj
5.3461 0 Td
(were)Tj
2.1203 0 Td
(collected)Tj
3.7418 0 Td
(for)Tj
1.3833 0 Td
(further)Tj
3.0273 0 Td
(analy-)Tj
-34.8545 -1.3039 Td
(sis.)Tj
1.4343 0 Td
(All)Tj
1.3946 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(above)Tj
2.5455 0 Td
(buffers)Tj
2.9877 0 Td
(were)Tj
2.126 0 Td
(made)Tj
2.4208 0 Td
(using)Tj
2.3924 0 Td
(DEPC)Tj
2.7496 0 Td
(treated)Tj
2.9934 0 Td
(water.)Tj
2.6588 0 Td
(Transcripts)Tj
4.7849 0 Td
(were)Tj
2.1203 0 Td
(extracted)Tj
-33.0914 -1.2982 Td
(using)Tj
2.3867 0 Td
(a)Tj
0.6576 0 Td
(phenol-chloroform)Tj
7.9087 0 Td
(procedure)Tj
4.3086 0 Td
([)Tj
0.83 0.64 0.02 0 k
(19)Tj
0 g
(].)Tj
2.0806 0 Td
(Before)Tj
2.812 0 Td
(RNA)Tj
2.262 0 Td
(precipitation)Tj
5.3631 0 Td
(in)Tj
1.0318 0 Td
(isopropanol,)Tj
5.2327 0 Td
(Vivid)Tj
-34.0438 -1.2983 Td
(Violet)Tj
2.6532 0 Td
(was)Tj
1.7007 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0262 0 Td
(samples)Tj
3.3845 0 Td
(as)Tj
1.0148 0 Td
(a)Tj
0.652 0 Td
(co-precipitating)Tj
6.599 0 Td
(factor)Tj
2.5455 0 Td
(\(E4502-01;)Tj
4.5581 0 Td
(EURx\).)Tj
3.1747 0 Td
(Precipitation)Tj
5.4028 0 Td
(was)Tj
-35.3704 -1.3039 Td
(performed)Tj
4.4673 0 Td
(at)Tj
0.9525 0 Td
(-20C)Tj
2.5795 0 Td
(for)Tj
1.3833 0 Td
(2)Tj
0.6973 0 Td
(hours.)Tj
2.7382 0 Td
(Purified)Tj
3.4526 0 Td
(RNA)Tj
2.262 0 Td
(was)Tj
1.7008 0 Td
(reverse)Tj
3.0444 0 Td
(transcribed)Tj
4.7565 0 Td
(to)Tj
1.0262 0 Td
(cDNA)Tj
2.8006 0 Td
(using)Tj
2.3867 0 Td
(random)Tj
-34.2479 -1.2983 Td
(hexamer)Tj
3.685 0 Td
(primers)Tj
3.3505 0 Td
(\(E0101-01;)Tj
4.5581 0 Td
(EURx\))Tj
2.9537 0 Td
(and)Tj
1.7291 0 Td
(MMLV)Tj
3.2371 0 Td
(reverse)Tj
3.0501 0 Td
(transcriptase)Tj
5.3291 0 Td
(\(M1701;)Tj
3.583 0 Td
(Promega\).)Tj
4.3653 0 Td
(Real)Tj
-35.841 -1.2983 Td
(time)Tj
2.0295 0 Td
(PCR)Tj
2.0637 0 Td
(analysis)Tj
3.3221 0 Td
(of)Tj
1.0148 0 Td
(mRNA)Tj
3.0841 0 Td
(was)Tj
1.7008 0 Td
(done)Tj
2.2223 0 Td
(using)Tj
2.3925 0 Td
(Real-Time)Tj
4.4163 0 Td
(2xPCR)Tj
3.0047 0 Td
(Master)Tj
2.9991 0 Td
(Mix)Tj
1.8481 0 Td
(\(2005100;)Tj
4.6318 0 Td
(A&A)Tj
-34.7298 -1.2982 Td
(Biotechnology\))Tj
6.3155 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
1.4684 0 Td
(Eco)Tj
1.7234 0 Td
(Real-Time)Tj
4.4164 0 Td
(PCR)Tj
2.0636 0 Td
(System)Tj
3.05 0 Td
(\(Illumina\).)Tj
4.5581 0 Td
(The)Tj
1.7802 0 Td
(following)Tj
3.9798 0 Td
(primers)Tj
3.3505 0 Td
(were)Tj
-34.435 -1.3039 Td
(used:)Tj
2.2847 0 Td
(C/EBP)Tj
/F11 1 Tf
()Tj
/F15 1 Tf
3.4072 0 Td
( )Tj
1.4003 0 Td
( D J F J D F J D J W D F D D J D W F F J)Tj
12.0245 0 Td
( )Tj
/F9 1 Tf
1.4003 0 Td
(and)Tj
/F15 1 Tf
1.7348 0 Td
( )Tj
1.4003 0 Td
( J F W J F W F F D F F W W F W W F W J F)Tj
12.0245 0 Td
( )Tj
/F9 1 Tf
(,)Tj
-35.6766 -1.2983 Td
(actin)Tj
/F15 1 Tf
2.1883 0 Td
( )Tj
1.4003 0 Td
( F D D J D J D W J J F F D F J J F)Tj
10.0402 0 Td
( W J F W W)Tj
3.1692 0 Td
( )Tj
/F9 1 Tf
1.4003 0 Td
(and)Tj
/F15 1 Tf
1.7291 0 Td
( )Tj
1.4003 0 Td
( F D J J W F W W W J F J J D W J W F)Tj
10.6298 0 Td
( F D F J)Tj
2.5796 0 Td
( )Tj
/F9 1 Tf
(,)Tj
1.627 0 Td
(28S)Tj
-36.1641 -1.2983 Td
(RNA)Tj
/F15 1 Tf
2.262 0 Td
( )Tj
1.4003 0 Td
( F D F F F D F W D D W D J J J D D F J W J)Tj
12.6141 0 Td
( )Tj
/F9 1 Tf
1.4003 0 Td
(and)Tj
/F15 1 Tf
1.7348 0 Td
( )Tj
1.4003 0 Td
( F W J D F W W D J D J J F J W W F D J W F)Tj
12.6141 0 Td
( )Tj
/F9 1 Tf
(.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 290.6645 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Western)Tj
3.9118 0 Td
(blotting)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 273.6566 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(For)Tj
1.627 0 Td
(western)Tj
3.3279 0 Td
(blotting)Tj
3.3562 0 Td
(analysis)Tj
3.3278 0 Td
(the)Tj
1.4684 0 Td
(following)Tj
3.9798 0 Td
(antibodies)Tj
4.3596 0 Td
(were)Tj
2.126 0 Td
(used:)Tj
2.2847 0 Td
(monoclonal)Tj
5.006 0 Td
(anti-FLAG)Tj
4.5864 0 Td
(M2)Tj
-35.4498 -1.2982 Td
(antibody)Tj
3.7643 0 Td
(1:1000)Tj
2.8233 0 Td
(\(F1804;)Tj
3.2145 0 Td
(SIGMA\))Tj
3.668 0 Td
(and)Tj
1.7291 0 Td
(peroxidase-conjugat)Tj
8.1127 0 Td
(ed)Tj
1.1679 0 Td
(goat)Tj
1.9275 0 Td
(anti-mouse)Tj
4.7565 0 Td
(antibody)Tj
-31.1638 -1.2983 Td
(1:10000)Tj
3.2995 0 Td
(\(554002;)Tj
3.6396 0 Td
(BD)Tj
1.5364 0 Td
(Pharmingen\).)Tj
5.7486 0 Td
(Immobilon)Tj
4.7792 0 Td
(transfer)Tj
3.3165 0 Td
(membrane)Tj
4.5694 0 Td
(\(IPVH00010;)Tj
5.5219 0 Td
(Millipore\))Tj
-32.4111 -1.2982 Td
(was)Tj
1.6951 0 Td
(incubated)Tj
4.1782 0 Td
(with)Tj
2.0012 0 Td
(Immobilon)Tj
4.7792 0 Td
(Western)Tj
3.6113 0 Td
(Chemiluminescent)Tj
7.8123 0 Td
(HRP)Tj
2.1656 0 Td
(Substrate)Tj
3.9175 0 Td
(\(WBKLS0500;)Tj
-30.1604 -1.304 Td
(Millipore\))Tj
4.2972 0 Td
(and)Tj
1.7292 0 Td
(visualized)Tj
4.1442 0 Td
(using)Tj
2.3868 0 Td
(the)Tj
1.474 0 Td
(ChemiDoc)Tj
4.5864 0 Td
(MP)Tj
1.6724 0 Td
(imaging)Tj
3.4866 0 Td
(system)Tj
2.9367 0 Td
(\(BioRad\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 194.9669 cm
BT
/F3 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Results)Tj
/F0 1 Tf
0 -1.5023 Td
(MCPIP1)Tj
3.9165 0 Td
(recognizes)Tj
4.9938 0 Td
(3'UTR)Tj
3.0236 0 Td
(of)Tj
1.0441 0 Td
(C/EBP)Tj
/F7 1 Tf
[()]TJ
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