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[(transfected)-494.8(with)-489.3(the)-488.4(plasmids)-494.9(using)-489.3(a)-486.1(Gene)-491.4(Pulser)-491.3(Xcell)]TJ
0 -1.1116 TD
[(electroporation)-286.6(system)-285.2(\(Bio-Rad)-282.8(Laboratories\).)]TJ
/F8 1 Tf
8.9663 0 0 8.9663 310.8472 725.7825 Tm
[(Flow)-332.3(cytometry)]TJ
/F3 1 Tf
9.843 0 0 9.843 320.8251 709.3416 Tm
[(Cell)-502.2(lines)-507.8(were)-508.7(harvested)-508.6(by)-503.3(brief)-507.8(exposure)-506.3(to)-509.2(0.25%)]TJ
-1.0137 -1.1116 TD
[(Trypsin/1)-166.7(mM)-338.4(EDTA)-342.6(\(Wako)-343(Pure)-339.4(Chemical)-343.1(Industries\).)]TJ
7.3823 0 0 6.5614 537.6188 702.7651 Tm
(\(22\))Tj
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[(After)-502.9(washing)-505(with)-500.9(phosphate-buffered)-508.1(saline)-504.3(\(PBS\),)-504.9(the)]TJ
T*
[(cells)-491.7(were)-497.1(treated)-495.2(with)-495.1(primary)-494(antibodies)-497.4(\(1)]TJ
/F4 1 Tf
19.5657 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(g/mL\))-497.6(for)]TJ
-20.1187 -1.1116 TD
[(30)-163.5(min)-419.4(at)-412.4(4)]TJ
/F10 1 Tf
4.7863 0 TD
()Tj
/F3 1 Tf
.3917 0 TD
[(C,)-414.5(follow)-7.7(ed)-412.3(by)-411.1(treatment)-418.2(with)-420.2(Oregon)-411.8(green-)]TJ
-5.178 -1.1174 TD
[(conjugated)-222.1(anti-mouse)-222(IgG)-217.2(\(Life)-221(Technologies\),)-224.1(Alexa)-222(Fluor)]TJ
0 -1.1116 TD
[(488)-245.2(conjugated)-250.9(anti-mouse)-250.8(IgG)-246(\(Cell)-249.8(Signaling)-247.5(Technology,)]TJ
T*
[(Danvers,)-348.2(MA\),)-348.3(or)-342(Alexa)-348.7(Fluor)-345.3(488)-343.1(conjugated)-348.8(anti-rat)-351.3(IgG)]TJ
T*
[(\(Cell)-434.2(Signaling)-431.8(Technology\).)-433.9(Fluorescence)-433.5(data)-433.1(were)-433.8(col-)]TJ
0 -1.1174 TD
[(lected)-563.3(using)-558.4(a)-561(FACS)-558.9(Calib)-8(ur)-555.1(ow)-562.7(cytometer)-562(\(BD)-563.9(Bio-)]TJ
0 -1.1116 TD
[(sciences,)-344.9(Braintree,)-340.6(MA\))-344.9(or)-336.2(a)-342.1(Cell)-340.9(Analyzer)-347.3(EC800)-341.8(\(Sony,)]TJ
T*
[(Tokyo,)-280.8(Japan\).)]TJ
/F8 1 Tf
8.9663 0 0 8.9663 310.8472 566.8723 Tm
[(Western)-336(blot)-331.8(analyses)]TJ
/F3 1 Tf
9.843 0 0 9.843 320.8251 550.4313 Tm
[(Cell)-456.1(lysates)-453.4(\(10)]TJ
/F4 1 Tf
6.8137 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(g\))-451.4(were)-456.8(boiled)-457.3(in)-451.6(SDS)-458.9(sample)-453.6(buffer)]TJ
-8.3804 -1.1116 TD
[(\(Nacalai)-314.1(Tesque,)-312.9(Kyoto,)-315.2(Japan\).)]TJ
7.3823 0 0 6.5614 439.7668 543.855 Tm
(\(33\))Tj
9.843 0 0 9.843 455.1306 539.4896 Tm
[(The)-316.7(proteins)-309.5(were)-312.8(elec-)]TJ
-14.6585 -1.1174 TD
[(trophoresed)-550(on)-555.1(520%)-548(polyacrylamide)-557.3(gels)-552.7(\(Wako)-550.3(Pure)]TJ
0 -1.1116 TD
[(Chemical)-521.7(Industries\))-525.7(and)-522.8(were)-525.9(transferred)-522.3(onto)-522.9(a)-520.6(PVDF)]TJ
T*
[(membrane)-325.8(\(EMD)-318.4(Millipore,)-326.9(Billerica,)-323.6(MA\).)-325.3(After)-324.4(blocking)]TJ
T*
[(with)-247.4(SuperBlock)-244.7(T20)-246.4(\(PBS\))-248.1(Blocking)-247.4(Buffer,)-248.3(the)-246.5(membrane)]TJ
0 -1.1174 TD
[(was)-321.9(incubated)-330.4(with)-322.3(primary)-327(antibodies)-324.6(\(1)]TJ
/F4 1 Tf
17.1524 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(g/mL\),)-328.3(and)-321.2(then)]TJ
-17.7053 -1.1116 TD
[(with)-621.8(peroxidase-conjugated)-629.3(secondary)-629.5(antibodies)-624.1(\(Dako,)]TJ
T*
[(Glostrup,)-255.1(Denmark;)-261(1:1000)-254.4(diluted\),)-261.4(and)-257.9(developed)-255.4(with)-259(the)]TJ
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[(ECL-plus)-219.5(reagent)-218.7(\(Thermo)-218.6(Fisher)-214.9(Scientic\))-221.9(using)-212.8(a)-221.1(Sayaca-)]TJ
0 -1.1116 TD
[(Imager)-280.8(\(DRC,)-281.5(Tokyo,)-280.8(Japan\).)]TJ
/F8 1 Tf
8.9663 0 0 8.9663 310.8472 418.9038 Tm
[(Immunohistochemical)-334(analyses)]TJ
/F3 1 Tf
9.843 0 0 9.843 320.8251 402.4629 Tm
(Four-)Tj
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2.2175 0 TD
(m)Tj
/F3 1 Tf
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[(m-thick)-344.4(histologic)-346.6(sections)-350.8(were)-347.4(deparafnized)-354.1(in)]TJ
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[(xylene)-279.8(and)-280.9(rehydrated.)-283(Then)-277.5(they)-282.2(were)-278.3(autoclaved)-286.6(in)-278.8(citrate)]TJ
T*
[(buffer)-359(\(pH)-361.2(6.0;)-358.2(Dako\))-358.9(for)-354.6(20)-169.2(min.)-359.4(Sections)-362.4(were)-358.9(incubated)]TJ
T*
[(with)-477.8(5)]TJ
/F4 1 Tf
2.9202 0 TD
(m)Tj
/F3 1 Tf
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[(g/mL)-479.3(of)-474.5(LpMab-3)-477.5(overnight)-477.8(at)-475.8(4)]TJ
/F10 1 Tf
14.2438 0 TD
()Tj
/F3 1 Tf
.3917 0 TD
[(C)-480.2(followed)-477.6(by)]TJ
-18.1085 -1.1174 TD
[(treatment)-354.8(with)-356.9(an)-348.9(Envision)]TJ
/F10 1 Tf
11.3178 0 TD
(+)Tj
/F3 1 Tf
1.0713 0 TD
[(kit)-354.9(\(Dako\).)-351.8(Color)-358(was)-350.7(devel-)]TJ
-12.3891 -1.1116 TD
[(oped)-316.6(using)-310.7(3,3-diaminobenzidine)-324.5(tetrahydrochloride)-318.7(\(DAB;)]TJ
T*
[(Dako\))-272.5(for)-268.2(10)-169.2(min,)-267.3(and)-269.4(the)-269.5(sections)-276(were)-272.5(counterstained)-273.7(with)]TJ
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[(hematoxylin)-281(\(Wako)-285.4(Pure)-281.8(Chemical)-285.5(Industries\).)]TJ
/F8 1 Tf
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[(Afnity)-334.2(determination)-332.9(by)-332.7(surface)-334.1(plasmon)-338.3(resonance)]TJ
/F3 1 Tf
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[(To)-222.3(determine)-228(the)-229.2(afnity,)-225.5(recombinant)-230.2(podoplanin-Fc)-229.8(was)]TJ
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[(immobilized)-374.5(on)-370.8(the)-373.2(surface)-373.8(of)-370.8(chips)-375.2(for)-371.9(analysis)-373.9(using)-374.1(the)]TJ
0 -1.1116 TD
[(BIAcore)-259.5(3000)-252.1(system)-256.4(\(GE)-258.8(Healthcare,)-257.5(Piscataway,)-261.8(NJ\).)-255.1(The)]TJ
T*
[(running)-555(buffer)-560.6(was)-558.1(10)-163.5(mM)-557.3(HEPES,)-559.1(150)-170.3(mM)-557.3(NaCl,)-558.2(and)]TJ
T*
[(0.005%)-632(v/v)-631.2(Surfactant)-630.8(P20)-631(\(BR-100368,)-633.9(pH)-630.9(7.4;)-634.7(GE)]TJ
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[(Healthcare\).)-759.7(LpMab-3)-753.9(was)-753.9(passed)-751.5(over)-754.3(the)-753.4(biosensor)]TJ
0 -1.1116 TD
[(chip,)-199.2(and)-200.3(the)-206.2(afnity)-199(rate)-202.7(constants)-202.2(\(association)-206.7(rate)-202.6(constant,)]TJ
/F9 1 Tf
T*
(k)Tj
/F3 1 Tf
7.3823 0 0 6.5614 315.2692 209.0834 Tm
(assoc)Tj
9.843 0 0 9.843 331.2566 210.7275 Tm
[(,)-199.3(and)-194.5(disassociation)-203.2(rate)-196.9(constant,)]TJ
/F9 1 Tf
13.4086 0 TD
(k)Tj
/F3 1 Tf
7.3823 0 0 6.5614 467.6597 209.0834 Tm
(diss)Tj
9.843 0 0 9.843 479.1117 210.7275 Tm
[(\))-196.9(were)-197.6(determined)]TJ
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[(by)-664.6(nonlinear)-674.8(curve-tting)-672.3(using)-667.8(the)-667(Langmuir)-674.9(one-site)]TJ
0 -1.1116 TD
[(binding)-376.6(model)-376.7(of)-370.8(the)-379(BIAevaluation)-377.2(software)-380.5(\(GE)-379.7(Health-)]TJ
T*
[(care\).)-369.5(The)-368.5(afnity)-366(constant)-368.3(\()]TJ
/F9 1 Tf
11.8592 0 TD
(K)Tj
/F3 1 Tf
7.3823 0 0 6.5614 434.1542 176.2016 Tm
(A)Tj
9.843 0 0 9.843 439.4266 177.8456 Tm
[(\))-369.7(a)0(t)-366.4(equilibrium)-366.3(was)-368(calcu-)]TJ
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[(lated)-221.2(as)]TJ
/F9 1 Tf
3.237 0 TD
(K)Tj
/F3 1 Tf
7.3823 0 0 6.5614 349.2282 165.2031 Tm
(A)Tj
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9.843 0 0 9.843 356.2015 166.8472 Tm
(=)Tj
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.72 0 TD
(k)Tj
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(assoc)Tj
9.843 0 0 9.843 383.6975 166.8472 Tm
(/)Tj
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(k)Tj
/F3 1 Tf
7.3823 0 0 6.5614 390.8976 165.2031 Tm
(diss)Tj
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[(,)-222.3(and)-223.3(the)-223.5(dissociate)-227.6(constant)-224.3(\()]TJ
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11.6749 0 TD
(K)Tj
/F3 1 Tf
7.3823 0 0 6.5614 523.8991 165.2031 Tm
(D)Tj
9.843 0 0 9.843 529.1715 166.8472 Tm
[(\))-225.7(was)]TJ
-22.1806 -1.1116 TD
[(determined)-280.9(as)-285.4(1/)]TJ
/F9 1 Tf
6.6928 0 TD
(K)Tj
/F3 1 Tf
7.3823 0 0 6.5614 383.3007 154.2614 Tm
(A)Tj
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(.)Tj
/F5 1 Tf
8.9663 0 0 8.9663 310.8472 133.285 Tm
(Results)Tj
/F8 1 Tf
0 -1.8336 TD
[(Production)-332(and)-335.7(characterization)-331.9(of)-336.7(novel)]TJ
0 -1.2203 TD
[(anti-podoplanin)-331.6(monoclonal)-337.8(antibody)-331.9(LpMab-3)]TJ
/F3 1 Tf
9.843 0 0 9.843 320.8251 89.4614 Tm
[(To)-325.9(develop)-332.7(novel)-329.3(anti-podoplanin)-336.1(MAbs,)-332(we)-326.8(immunized)]TJ
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[(mice)-491.9(with)-489.3(LN229/hPDPN)-493.8(cells.)-489.4(The)-495.2(culture)-488.3(supernatants)]TJ
0 -1.1174 TD
[(were)-543.2(screened)-548.9(using)-541.1(ELISA)-550(for)-544.6(binding)-543.6(to)-538(recombinant)]TJ
0 -1.1116 TD
[(human)-272.8(podoplanin)-271.6(puried)-273.8(from)-270.5(LN229/hPDPN)-274.9(cells.)-270.5(After)]TJ
/F5 1 Tf
8.9663 0 0 8.9663 59.7543 749.7069 Tm
[(CHARACTERIZATION)-341.2(OF)-337.5(LpMab-3)-333(ANTI-PDPN)-340.8(Mab)-28964.3(45)]TJ
ET
endstream
endobj
52 0 obj
<>stream
/GS1 gs
BT
/F3 1 Tf
9.843 0 0 9.843 62.022 725.1588 Tm
0 0 0 rg
0 Tc
0 Tw
[(limiting)-488.7(the)-488.4(dilution)-493.1(of)-486(the)-488.4(hybridomas,)-489(LpMab-3)-494.8(\(IgG)]TJ
7.3823 0 0 6.5614 294.9731 723.5148 Tm
(1)Tj
9.843 0 0 9.843 298.6582 725.1588 Tm
(,)Tj
-24.041 -1.1174 TD
[(kappa\))-625.2(was)-621.4(established.)-623.8(LpMab-3)-621.5(reacted)-623(with)-621.8(LN229/)]TJ
0 -1.1116 TD
[(hPDPN,)-459.6(not)-452.7(with)-454.8(LN229,)-460.3(a)-457.3(podoplanin-negative)-458.1(cell)-456.2(line)]TJ
T*
[(\(Fig.)-341.8(1A\).)-347.4(Furthermore,)-347.3(LpMab-3)-345(detected)-347.8(endogenous)-347.2(po-)]TJ
T*
[(doplanin,)-307.4(which)-301.4(is)-307.4(expressed)-306.7(in)-301.8(LN319)-307.1(\(a)-302.8(glioblastoma)-305.1(cell)]TJ
25.504 4.4523 TD
[(line\),)-315.6(a)-313.3(lymphatic)-313.4(endothelial)-315.6(cell)-312.3(\(LEC\),)-311.8(and)-315.5(NCI-H226)-311(\(a)]TJ
0 -1.1174 TD
[(malignant)-371(mesothelioma)-370.7(cell)-369.9(line\))-364(\(Fig.)-370.6(1B\).)-364.8(We)-368.5(next)-368.6(per-)]TJ
0 -1.1116 TD
[(forme)-7.8(d)-519.5(ow)-522.4(cytometric)-528.7(analyses)-524.7(using)-523.8(LpMab-3)-523.6(against)]TJ
T*
[(several)-350.9(glycan-decient)-350.8(podoplanin)-352.3(transfectants)-347.2(\(Fig.)-347.6(1C\).)]TJ
T*
[(LpMab-7,)-250.5(which)-243.8(was)-241.3(used)-247.3(as)-245.1(a)-244.2(positive)-246.3(control,)-242.9(reacted)-248.6(with)]TJ
ET
q
1 i
67.918 656.107 m
546.236 656.107 l
546.236 163.332 l
67.918 163.332 l
W n
q
478.481 0 0 492.775 67.918 163.332 cm
/Im1 Do
Q
Q
BT
/F6 1 Tf
9.845 0 0 9.845 62.022 146.6078 Tm
[(FIG.)-446.2(1.)]TJ
/F3 1 Tf
4.2268 0 TD
(\()Tj
/F6 1 Tf
.334 0 TD
(A)Tj
/F3 1 Tf
.7198 0 TD
[(\))-444.4(Flow)-448.2(cytometric)-447(analysis)-447.9(by)-445.4(LpMab-3)-447.7(against)-452.9(LN229/hPDPN)-446.2(and)-447.6(LN229.)-447.9(Cell)-444.1(lines)-449.7(were)-450.5(treated)-448.4(with)]TJ
-5.2806 -1.0135 TD
[(LpMab-3)-246.2(\()16.3(1)]TJ
/F4 1 Tf
4.9927 0 TD
(m)Tj
/F3 1 Tf
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-.0161 Tc
[(g/)-16.1(m)9.4(L)-1(\))-241.7(for)-247.4(30)-166.8(min)-241.7(a)-1(t)-241.8(4)]TJ
/F10 1 Tf
8.3614 0 TD
0 Tc
()Tj
/F3 1 Tf
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-.0158 Tc
[(C,)-243.8(follo)-15.8(w)10.2(e)-.7(d)-241.4(b).5(y)-241.4(t)-15.8(r)10.8(eatmen)-15.8(t)-231(w)-.3(ith)-247.1(Oregon)-247.1(green-)-15.8(c)9.8(onju)-15.8(g)11(ated)-241.4(a)-15.8(n)9.8(ti-mo)-15.8(u)11(s).7(e)-242.6(I).5(g)-15.8(G)10.2(.)-243.8(F).7(luor)-15.8(e)9.8(s).6(cen)-15.8(c)9.8(e)-242.5(data)]TJ
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[(wer)-15.7(e)-329.8(col)-15.7(l)10.8(ected)-344.9(usin)-15.7(g)-328.6(a)-340.3(F)-15.7(A)10.5(CS)-339(Ca)-15.7(l)9.8(i).4(bur)-344.9(ow)-345.8(c)-.6(ytomet)-15.7(e)9.8(r).6(.)-341.6(\()]TJ
/F6 1 Tf
22.1301 0 TD
0 Tc
(B)Tj
/F3 1 Tf
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[(\))-345(W)-1.7(este)-15.8(r)9.8(n)-339.3(bl)-15.8(o)10.8(t)-339.5(ana)-15.8(l)9.7(ysi)-15.8(s)-328.7(by)-345(LpMab-)-15.8(3)11(.)-341.7(Total)-345.2(cell)-345.2(l).3(ysat)-15.8(e)-330.1(w)-.3(ere)-346.2(e)-.7(lect)-15.8(r)10.8(o).5(-)]TJ
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[(pho)-15.8(r)11(e)-.7(sed)-264.4(o).5(n)-258.7(5).5()-15.8(2)11(0%)-259.6(po)-15.8(l)10.8(y).5(acr)-15.8(y)11(l).3(amide)-265.6(gels)-264.2(and)-258.7(t).3(r)-15.8(a)9.8(nsf)-15.8(e)9.8(rred)-264.4(onto)-264.4(a)-259.8(PVDF)-264.2(membran)-15.8(e)9.8(.)-261(A)-.3(fter)-264.4(bloc)-15.8(k)9.8(i).3(ng,)-261(th)-15.8(e)-249.3(membra)-15.8(n)9.8(e)-259.8(was)-264.2(i).3(ncu)-15.8(b)11(ated)]TJ
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[(w)15.5(i)16.1(th)-284.3(1)]TJ
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(m)Tj
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[(Ima)-15.7(g)9.9(er.)-404.9(\()]TJ
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(C)Tj
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[(li)-15.8(n)10.8(e)-.7(s.)-261(Cell)-258.8(l)-15.8(i)10.7(nes)-258.5(w)-15.8(e)9(r).5(e)-259.8(t).3(r)-15.8(e)9.8(ated)-258.7(wi)-15.8(t)10.7(h)-258.7(LpMa)-15.8(b)9.8(-).5(3)-258.7(a)-.7(nd)-264.4(LpMab-)-15.8(7)-248.1(\().5(1)]TJ
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(m)Tj
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[(g/)-16.1(m)9.4(L)-1(\))-259(for)-264.7(30)-166.8(min)-259(a)-1(t)-264.9(4)]TJ
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()Tj
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[(C,)-261(follo)-15.8(w)10.2(e)-.7(d)-258.7(b).5(y)-258.7(t)-15.8(r)10.8(eatme)-15.8(n)9.8(t)-258.8(with)-264.4(Alexa)-259.8(F)-15.8(l)11(uor)]TJ
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