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0 -1.1116 TD
[(tor)-9.2(ies,)-238.2(Inc.,)-241.7(Burlin)-7.9(game,)-241.8(CA\))-235.6(for)-239.4(30)-238.3(minut)-7(es.)-237(The)-241.8(membrane)]TJ
T*
[(wa)13(s)-317.7()0(n)21(a)0(l)19.5(l)0(y)-307.6(d)0(e)13.9(v)0(e)19.7(l)0(o)20.7(p)0(e)19.7(d)-317.9(wi)19.8(th)-313.3(th)20.7(e)-319(I)0(m)19.6(m)0(u)19.6(n)0(o)20.8(S)0(t)20.9(a)0(r)-314.4(L)0(D)-309.5(C)0(h)19.8(e)0(m)18.5(i)0(l)20.6(u)0(m)19.6(i)0(-)]TJ
0 -1.1174 TD
.0198 Tc
[(nesc)-5.5(ence)-316.5(R)-5.4(eage)-5.5(n)1.4(t)-315.5(\()1.5(FUJIFILM)-316.3(Wa)-5.5(ko)-315.4(Pure)-316.5(Chem)-5.6(ical)-315.5(Cor-)]TJ
0 -1.1116 TD
0 Tc
[(poration,)-267.1(Osaka,)-271.1(Japan\))-273.7(using)-264.6(the)-275.3(Sayaca-Im)7.5(ager)-271.6(\(DRC)-272.3(Co.,)]TJ
T*
[(Ltd.,)-253.4(Tokyo,)-246.3(Japan\).)-248.3(All)-246.3(procedures)-250.4(of)-249.8(western)-247(blotting)-245.5(were)]TJ
0 -1.1174 TD
[(performed)-278.3(at)-280(the)-275.3(room)-276.3(temperature.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 441.9778 Tm
[(Immunocytochemical)-338.4(analysis)]TJ
/F4 1 Tf
9.843 0 0 9.843 323.0361 425.5369 Tm
[(HeLa)-221.9(cells)-226.8(were)-220.7(xed)-218.5(with)-224.4(2%)-216.4(paraformaldehyde)-226.3(in)-221.2(0.1)-166.9(M)]TJ
-1.0137 -1.1116 TD
[(phosph)-6.4(ate)-293.7(buffer)-295.7(\(pH)-292.1(7.4\))-288.9(for)-297(20)-290.2(minutes)-298.2(on)-290.2(ice.)-297.2(After)-295.6(xa-)]TJ
T*
[(tion,)-307.6(cells)-301.6(were)-307.1(perforated)-302.5(with)-305(0.3%)-301.5(Triton-X)-304.8(100/PBS)-302.3(for)]TJ
0 -1.1174 TD
[(30)-261.4(minutes)-263.6(at)-256.9(room)-259(temperature,)-263.5(followed)-264.5(by)-261.4(treatment)-262.7(with)]TJ
0 -1.1116 TD
[(5%)-187.6(bovine)-186.5(serum)-186.2(albumin)-188.9(\(BSA\))-185.7(in)-186.6(PBS)-188.3(\(BSA/PBS\))-185.2(to)-186.6(block)]TJ
T*
[(nonspecic)-244.7(binding)-238.4(sites,)-234.6(and)-240.6(were)-238(incubated)-238.3(with)-241.7(10)]TJ
/F2 1 Tf
21.5643 0 TD
(m)Tj
/F4 1 Tf
.5529 0 TD
(g/mL)Tj
-22.1173 -1.1174 TD
[(of)-434.1(DzMab-1)-432.2(or)-428.4(control)-429.6(\(BSA/PBS\))-438.7(for)-429.5(overnight)-431.8(at)-429.7(room)]TJ
0 -1.1116 TD
[(temperatur)-10.2(e)-324.8(i)0(n)-330.6(a)-324.8(moist)-329.4(chamber.)-332.6(They)-329.3(were)-330.1(incubated)-336.2(with)]TJ
T*
[(goat)-316.7(anti-rat)-311(IgG-Alexa)-319.3(488)-308.6(\(dilution)-309.9(1:300;)-312.1(Thermo)-315.4(Fisher)]TJ
0 -1.1174 TD
[(Scientic,)-391.3(Inc.\))-389.1(for)-383.4(30)-388.1(minutes)-384.6(at)-389.4(room)-385.7(temperature.)-390.2(Cells)]TJ
0 -1.1116 TD
[(were)-249.5(also)-247.4(treated)-253.3(with)-247.4(DAPI)-247.9(\(Thermo)-247.4(Fisher)-249.4(Scientic,)-247.3(Inc.\))]TJ
T*
[(to)-301.8(stain)-294.7(the)-304.1(cell)-295(nuclei.)-300.6(They)-300.5(were)-301.3(examined)-301.6(using)-293.4(confocal)]TJ
T*
[(laser)-301.5(scanning)-299.1(microscopy)-302.5(\(LSM700;)-298.8(Carl)-300.4(Zeiss,)-299(Inc.,)-299.3(Jena,)]TJ
0 -1.1174 TD
(Germany\).)Tj
/F6 1 Tf
8.9663 0 0 8.9663 313.0582 259.3133 Tm
[(Results)-337.2(and)-331.9(Discussion)]TJ
/F4 1 Tf
9.843 0 0 9.843 323.0361 242.8724 Tm
[(Previously,)-637.5(immunohistochemical)-640.3(studies)-630.8(using)-633.3(rabbit)]TJ
-1.0137 -1.1116 TD
[(polyclonal)-359.3(DGK)]TJ
/F2 1 Tf
6.7561 0 TD
(z)Tj
/F4 1 Tf
.7891 0 TD
[(antibody)-361.6(on)-353.5(rat)-355.9(tissues)-359.9(showed)-354(predomi-)]TJ
-7.5452 -1.1174 TD
[(nant)-420.4(localization)-427.5(of)-416.9(the)-425.1(immunoreactivity)-421.6(in)-422.8(the)-419.3(nuclei)-418.1(of)]TJ
0 -1.1116 TD
[(various)-354.4(cell)-346.8(types,)-349.9(including)-345.5(neurons,)-351.9(hepatocytes,)-348.6(alveolar)]TJ
T*
[(epithelial)-320.4(cells,)-310.8(and)-315.5(macrophages.)]TJ
7.3823 0 0 6.5614 448.6109 203.4141 Tm
(\(6\))Tj
9.843 0 0 9.843 460.3463 199.0488 Tm
[(Nuclear)-311.7(localization)-318(of)]TJ
-14.9637 -1.1174 TD
(DGK)Tj
/F2 1 Tf
2.1599 0 TD
(z)Tj
/F4 1 Tf
.6278 0 TD
[(immunoreactivity)-191.2(is)-192.2(consistent)-191.9(with)-189.8(cDNA)-192.5(tranfection)]TJ
-2.7877 -1.1116 TD
[(studie)-7.8(s)-277.4(i)0(n)-278.8(COS-7)-283.7(cells)-284.4(and)-280.9(primary)-280.9(cultured)-282.1(neurons.)]TJ
7.3823 0 0 6.5614 524.466 181.474 Tm
(\(10,14\))Tj
9.843 0 0 9.843 323.0361 166.1669 Tm
[(As)-319.7(this)-321.3(antibody)-315.5(was)-321.9(not)-320.2(useful)-318.7(in)-319.1(immunohistochemical)]TJ
-1.0137 -1.1116 TD
[(study)-362.5(on)-359.3(human)-359.2(tissues)-365.7(and)-355.8(cells,)-362.7(we)-361.3(aimed)-366.3(at)-354.8(obtaining)-362.8(a)]TJ
0 -1.1174 TD
[(specic)-310.5(DGK)]TJ
/F2 1 Tf
5.5293 0 TD
(z)Tj
/F4 1 Tf
.7372 0 TD
[(antibody)-309.7(that)-305.3(is)-307.4(suitable)-305.1(for)-308.5(immunostaining)]TJ
-6.2666 -1.1116 TD
[(of)-451.4(human)-445.6(cells.)-454.8(The)-449.2(recombinant)-449.1(maltose-binding)-452.4(protein)]TJ
T*
[(\(MBP\)-)-8.3(mDGK)]TJ
/F2 1 Tf
6.0477 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-194(was)-195.2(captured)-196.7(using)-189.8(NZ-1-Sephar)-10.5(ose)-194.3(and)]TJ
-6.4854 -1.1174 TD
[(efciently)-331.5(eluted)-326(with)-328.1(PA)-319.7(tag)-327.1(peptide.)-324.8(Rat)-328.2(was)-327.7(then)-322.5(immu-)]TJ
0 -1.1116 TD
[(nized)-255.6(with)-253.2(MBP-mDGK)]TJ
/F2 1 Tf
9.8376 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-257.4(to)-250(develop)-252.1(novel)-254.5(anti-mDGK)]TJ
/F2 1 Tf
13.5814 0 TD
(z)Tj
/F4 1 Tf
-23.8567 -1.1116 TD
[(mAbs.)-234.4(Using)-225.2(ELISA,)-230.9(the)-229.2(culture)-229.1(supernatants)-235.4(of)-226.8(hybridomas)]TJ
0 -1.1174 TD
[(were)-272.5(screened)-266.7(for)-262.4(the)-269.5(binding)-261.4(to)-267.3(MBP-mDGK)]TJ
/F2 1 Tf
18.6614 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-263.2(that)-270.8(was)]TJ
-19.0992 -1.1116 TD
[(puried)-446.5(from)]TJ
/F10 1 Tf
5.9498 0 TD
[(E.)-443.5(coli)]TJ
/F4 1 Tf
2.8165 0 TD
[(.)-441.2(Furthermore)-9.8(,)-441.2(MBP-hDGK)]TJ
/F2 1 Tf
11.4906 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-441.7(was)]TJ
-20.6946 -1.1116 TD
[(used)-224.2(as)-222(a)-221.1(second)-221.8(ELISA)-227.4(screening.)-224.1(As)-221.7(a)-221.1(result,)-224.3(DzMab-1)-224.9(\(rat)]TJ
/F6 1 Tf
8.9663 0 0 8.9663 62.022 749.6502 Tm
(180)Tj
46.7326 0 TD
[(NAKANO)-333.3(ET)-338.7(AL.)]TJ
ET
endstream
endobj
29 0 obj
<>stream
/GS1 gs
BT
/F4 1 Tf
9.843 0 0 9.843 59.7543 549.2408 Tm
0 0 0 rg
0 Tc
0 Tw
(IgG)Tj
7.3823 0 0 6.5614 75.0614 547.5967 Tm
(1)Tj
9.843 0 0 9.843 78.7464 549.2408 Tm
[(,)-504.5(kappa\))-510(that)-506.9(can)-506.7(react)-510.2(with)-506.6(both)-505.6(MBP-mDGK)]TJ
/F2 1 Tf
20.3202 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
(-PA)Tj
-22.6875 -1.1116 TD
[(and)-465.2(MBP-hDGK)]TJ
/F2 1 Tf
7.0038 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-464.7(was)-465.9(established)-464.9(after)-462.9(limiting)-465.7(dilu-)]TJ
-7.4415 -1.1116 TD
[(tion.)-601.3(Western)-605.5(blot)-597.9(analysis)-598.5(demonstrated)-605.5(that)-599.1(DzMab-1)]TJ
0 -1.1174 TD
[(detected)-635.8(MBP-hDGK)]TJ
/F2 1 Tf
9.0831 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
[(-PA)-631.8(but)-631.2(not)-631.2(MBP-hDGK)]TJ
/F2 1 Tf
11.1508 0 TD
(a)Tj
/F4 1 Tf
.5529 0 TD
[(-PA)-631.8(or)]TJ
-21.2245 -1.1116 TD
(MBP-hDGK)Tj
/F2 1 Tf
5.0973 0 TD
(g)Tj
/F4 1 Tf
.5011 0 TD
[(-PA)-297.7(\(Fig.)-295.8(1\),)-299.3(indicating)-296.1(that)-299.6(DzMab-1)-299.8(speci-)]TJ
-5.5984 -1.1116 TD
[(cally)-284.6(reacts)-286.5(with)-282(hDGK)]TJ
/F2 1 Tf
9.5842 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
(.)Tj
-9.0082 -1.1174 TD
[(In)-318.9(this)-315.5(study,)-319.9(we)-315.3(used)-316.4(HeLa)-319.8(cells,)-322.4(a)-313.3(human)-318.9(cervical)-324.7(can-)]TJ
-1.0137 -1.1116 TD
[(cer)-431.8(cell)-433.2(line,)-429.7(in)-428.5(which)-428.1(expression)-433.5(of)-428.4(DGK)]TJ
/F2 1 Tf
17.9472 0 TD
(z)Tj
/F4 1 Tf
.864 0 TD
[(protein)-429.6(is)-428.3(re-)]TJ
-18.8112 -1.1116 TD
(ported.)Tj
7.3823 0 0 6.5614 87.4771 465.9589 Tm
(\(15,16\))Tj
9.843 0 0 9.843 114.9165 461.5936 Tm
[(Immunocytochemical)-599.9(analysis)-598.5(revealed)-601(that)]TJ
-5.6042 -1.1174 TD
[(immunoreactivity)-461.9(for)-452.5(DzMab-1)-461(is)-451.4(predominantly)-456(detected)]TJ
0 -1.1116 TD
[(in)-388.2(the)-379(nucle)-8(us)-382.1(with)-385.7(a)-382.4(granular)-385.6(pattern)-384.6(of)-382.3(labeling)-391.7(and)-378.8(less)]TJ
T*
[(intensely)-421.4(in)-411.3(the)-419.3(cytoplasm)-416.7(with)-414.5(a)-417(diffuse)-411.9(pattern)-419.2(\(Fig.)-416.7(2\).)]TJ
T*
[(Immunoreactivity)-248.7(observed)-241.3(in)-244.2(this)-240.6(study)-241.6(is)-238.3(similar)-246.4(to)-238.5(the)-240.7(one)]TJ
0 -1.1174 TD
[(in)-284.6(previous)-280.6(studies)-285.2(using)-276.1(rabbit)-284.5(polyclonal)-284.4(antibody.)]TJ
7.3823 0 0 6.5614 268.4976 411.1369 Tm
(\(17,18\))Tj
9.843 0 0 9.843 320.8251 725.1588 Tm
[(Immunostaining)-376.3(without)-377.6(primary)-378.8(antibody)-378.9(abolished)-380.9(the)]TJ
-1.0137 -1.1174 TD
[(immunoreactions)-471.9(\(data)-468.7(not)-464.2(shown\).)-471.5(Furthermore,)-468.2(we)-470.8(per-)]TJ
0 -1.1116 TD
[(formed)-343(siRNA)-348(silencing)-346.5(experiment.)-347.7(As)-342.7(given)-346.6(in)-342.1(Figure)-347.5(3,)]TJ
T*
[(transfection)-466.1(with)-466.3(20)-169.2(pmol)-460.8(of)-468.7(siRNA)-463.2(for)-464(DGK)]TJ
/F2 1 Tf
19.462 0 TD
(z)Tj
/F4 1 Tf
.8985 0 TD
(\(siDGK)Tj
/F2 1 Tf
3.1563 0 TD
(z)Tj
/F4 1 Tf
.4377 0 TD
(\))Tj
-23.9546 -1.1116 TD
[(signicantly)-557.1(reduced)-558.3(the)-557.5(immunoreactivity)-565.6(for)-556.2(DzMab-1)]TJ
0 -1.1174 TD
[(compared)-226.6(with)-224.4(the)-223.5(transfection)-229.9(with)-224.4(scramble)-226.5(control)-222.2(siRNA)]TJ
0 -1.1116 TD
[(\(siControl\).)-293.1(These)-296.8(results)-296.7(conrm)-296.8(that)-293.8(DzMab-1)-294(reacts)-298(with)]TJ
T*
[(native)-349(DGK)]TJ
/F2 1 Tf
4.9649 0 TD
(z)Tj
/F4 1 Tf
.7833 0 TD
[(protein)-348.9(specically)-356.6(in)-347.9(human)-353.5(HeLa)-348.6(cells)-353.5(and)]TJ
-5.7482 -1.1174 TD
[(that)-282.3(the)-281.1(images)-280.8(obtained)-282.1(with)-282(this)-280.9(antibody)-280.9(is)-284.4(authentic.)]TJ
1.0137 -1.1116 TD
[(In)-221(addition,)-221.2(it)-227.1(should)-219.7(be)-228(mentioned)-221.1(that)-224.7(many)-225.7(of)-221(the)-223.5(nuclei)]TJ
-1.0137 -1.1116 TD
[(in)-370.9(cells)-382.3(transfected)-379.6(with)-374.2(40)-169.2(pmol)-374.4(of)-376.5(siDGK)]TJ
/F2 1 Tf
17.9472 0 TD
(z)Tj
/F4 1 Tf
.8121 0 TD
[(are)-374.2(shrunk)-380.8(or)]TJ
-18.7594 -1.1174 TD
[(irregular)-242.8(\(Fig.)-243.9(3,)-240.7(lower)-238(panel)-8(s\).)-241.6(Nuclear)-242.6(deformation)-246.3(is)-238.3(more)]TJ
0 -1.1116 TD
[(evident)-402(in)-394(cells)-405.3(transfected)-402.7(with)-397.2(60)-169.2(pmol)-397.4(of)-399.6(siDGK)]TJ
/F2 1 Tf
21.4434 0 TD
(z)Tj
/F4 1 Tf
.8352 0 TD
(\(data)Tj
-22.2785 -1.1116 TD
[(not)-446.9(shown\).)-442.7(No)-446.6(changes)-448.7(in)-445.8(the)-448.1(nuclear)-449(morphology)-444.3(were)]TJ
ET
q
154.038 0 0 84.813 102.444 647.433 cm
/Im1 Do
Q
BT
/F5 1 Tf
9.845 0 0 9.845 59.7543 630.6518 Tm
[(FIG.)-636.2(1.)]TJ
/F4 1 Tf
4.4168 0 TD
[(Specicity)-641.3(of)-635.4(DzMab-1)-632.8(for)-636.4(DGK)]TJ
/F2 1 Tf
14.8225 0 TD
(z)Tj
/F4 1 Tf
.4376 0 TD
[(.)-631.1(Immuno-)]TJ
-19.6769 -1.0135 TD
[(precipitates)-264.7(of)-261.1(hDGK)]TJ
/F2 1 Tf
8.6436 0 TD
(a)Tj
/F4 1 Tf
.5528 0 TD
[(,)-256.8(hDGK)]TJ
/F2 1 Tf
3.1672 0 TD
(g)Tj
/F4 1 Tf
.4952 0 TD
[(,)-262.5(and)-257.5(hDGK)]TJ
/F2 1 Tf
4.8717 0 TD
(z)Tj
/F4 1 Tf
.6968 0 TD
[(by)-261.1(anti-PA)-256.7(tag)]TJ
-18.4273 -1.0135 TD
[(\(NZ-1\))-543.5(were)-542.7(electrophoresed)-544.9(and)-539.7(transferred)-544.2(onto)-539.7(mem-)]TJ
0 -1.0077 TD
[(branes.)-299.4(After)-295(blocking,)-295(the)-298(membranes)-296.7(were)-295.1(incubated)-300.7(with)]TJ
0 -1.0135 TD
(10)Tj
/F2 1 Tf
1.169 0 TD
(m)Tj
/F4 1 Tf
.5528 0 TD
[(g/mL)-363.5(of)-359(DzMab-1)-362.2(and)-361.2(1)]TJ
/F2 1 Tf
10.3999 0 TD
(m)Tj
/F4 1 Tf
.5528 0 TD
[(g/mL)-363.5(of)-359(NZ-1,)-366.4(respectively.)]TJ
-12.6746 -1.0135 TD
[(hDGK,)-251.6(human)-249.1(diacylglycerol)-249.6(kinase;)-251.3(PA,)-247.9(phosphatidic)-250.7(acid.)]TJ
ET
q
1 i
60.321 381.543 m
298.261 381.543 l
298.261 123.137 l
60.321 123.137 l
W n
q
238.023 0 0 258.406 60.321 123.137 cm
/Im2 Do
Q
Q
BT
/F5 1 Tf
9.845 0 0 9.845 59.7543 106.4126 Tm
[(FIG.)-486.5(2.)]TJ
/F4 1 Tf
4.2671 0 TD
[(Immunocytochemistry)-486.8(using)-482.9(DzMab-1)-483.1(in)-485.9(HeLa)]TJ
-4.2671 -1.0135 TD
[(cells.)-339.1(HeLa)-342.3(cells)-335.7(were)-341.1(stained)-337.7(with)-339.1(DzMab-1)-339.1(antibody)-337.7(at)-337.3(a)]TJ
T*
[(concentration)-300.2(of)-289.9(10)]TJ
/F2 1 Tf
8.0562 0 TD
(m)Tj
/F4 1 Tf
.5528 0 TD
[(g/mL.)-292(Nuclear)-299.4(staining)-291.6(was)-298.5(performed)]TJ
-8.609 -1.0077 TD
[(with)-333.4(DAPI.)-337(Enlarged)-334.1(view)-334.4(\(lower)-336.3(left)-333.7(panel\))-336.7(clearly)-339(shows)]TJ
0 -1.0135 TD
[(granular)-350.2(staining)-349.2(in)-347.7(the)-349.8(nucleus)-349(\(N\))-343.6(and)-349.7(diffuse)-347.9(staining)-349.2(in)]TJ
T*
[(the)-338.3(cytoplasm.)-332.7(Bar)]TJ
/F8 1 Tf
7.8892 0 TD
(=)Tj
/F4 1 Tf
.7141 0 TD
(10)Tj
/F2 1 Tf
1.169 0 TD
(m)Tj
/F4 1 Tf
.5528 0 TD
(m.)Tj
ET
q
1 i
311.414 548.844 m
549.354 548.844 l
549.354 143.093 l
311.414 143.093 l
W n
q
238.023 0 0 405.751 311.414 143.093 cm
/Im3 Do
Q
Q
BT
/F5 1 Tf
9.845 0 0 9.845 310.8472 126.3118 Tm
[(FIG.)-607.4(3.)]TJ
/F4 1 Tf
4.388 0 TD
[(Knockdown)-604.8(of)-606.6(DGK)]TJ
/F2 1 Tf
9.087 0 TD
(z)Tj
/F4 1 Tf
1.0423 0 TD
[(using)-609.6(siRNA)-606.5(in)-606.8(HeLa)]TJ
-14.5173 -1.0077 TD
[(cells.)-356.4(Cells)-364.3(were)-358.4(transfected)-361.3(with)-356.4(siDGK)]TJ
/F2 1 Tf
16.8956 0 TD
(z)Tj
/F4 1 Tf
.7947 0 TD
[(at)-360.4(an)-360.2(amount)-359.7(of)]TJ
-17.6902 -1.0135 TD
[(20)-163.2(pmol)-598.4(\(upper)-600.2(panels\))-596.6(and)-597.3(40)-169(pmol/60)-163.9(mm)-597.4(dish)-597.1(\(lower)]TJ
T*
[(panels\).)-559.7(After)-565.7(48)-560.6(hours,)-561(immunocytochemistry)-561.9(was)-563.4(per-)]TJ
T*
[(formed)-278.9(using)-275.6(DzMab-1.)-279.2(Note)-276.8(that)-281.9(shrunk)-276.4(or)-278.4(irregular)-276.5(nuclei)]TJ
0 -1.0077 TD
[(are)-523.6(frequently)-525.1(observed)-522.6(in)-520.4(40)-169(pmol)-523.6(siDGK)]TJ
/F2 1 Tf
18.4101 0 TD
(z)Tj
/F4 1 Tf
.4376 0 TD
[(-treated)-524.3(cells)]TJ
-18.8477 -1.0135 TD
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[(used)-333.2(as)-337(a)-330.4(control.)-334.3(Bar)]TJ
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