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[(R)-6.5(A)-.5(P-)-5.4(MAP)-5.3(/).2(pCA)-6.3(G)-.5(,)-1079.1(a)-.8(nd)-1076.7(P)-5.3(A)-.5(-C)-6.5(D13)-5.4(3).3(-RA)-6.3(P).5(-M)-6.3(AP/)-5.6(p).3(CAG)-6.3(,)]TJ
0 -1.1174 TD
0 Tc
[(respectivel)-11.2(y\).)-420.3(The)-420.4(MAP-tagged)-429.8(deletion)-420.5(mutant)-424(of)-422.6(human)]TJ
0 -1.1116 TD
[(podoplanin)-288.9(without)-285.4(the)-286.8(PLAG)-289.4(domain)-285.6(\()-81.7(MAP-hPDPNdN)-10.2(55/)]TJ
T*
[(pCAG\))-330.8(was)-327.7(cloned)-325.8(into)-327.2(the)-327.1(pCAG)-324(vector)-331.6(as)-325.7(described)-330.1(pre-)]TJ
T*
(viously.)Tj
7.3823 0 0 6.5614 344.5794 685.7006 Tm
(\(20\))Tj
9.843 0 0 9.843 359.0928 681.3352 Tm
[(All)-223.3(constructs)-232(were)-226.4(veried)-228.8(by)-221.1(DNA)-224.8(sequencing.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 659.3951 Tm
[(Binding)-338.3(kinetics)-333.7(analysis)-334.5(using)-332.2(surface)]TJ
0 -1.2267 TD
[(plasmon)-338.3(resonance)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 631.9557 Tm
[(Rat)-506.7(anti-MAP)-507.1(mAb)-506.5(PMab-1)-509.6(\(IgG)]TJ
7.3823 0 0 6.5614 465.9589 630.3116 Tm
(2a)Tj
9.843 0 0 9.843 472.9889 631.9557 Tm
(,)Tj
/F10 1 Tf
.7545 0 TD
(k)Tj
/F3 1 Tf
.5529 0 TD
[(\))-502.2(was)-512(described)]TJ
-17.5556 -1.1116 TD
[(pr)15.1(ev)19.7(io)20.7(us)21(ly)20.7(.)]TJ
7.3823 0 0 6.5614 356.0881 625.3794 Tm
[(\(1)26.6(5\))]TJ
9.843 0 0 9.843 370.0912 621.014 Tm
[(PM)20.1(ab)13.9(-1)-192.2(wa)18.8(s)-202.5(i)0(m)19.5(m)0(o)19.6(b)0(i)20.7(l)0(i)20.6(z)0(e)18.5(d)-202.7(on)-192.3(CM)18.9(5)-202.7(s)0(e)19.9(n)0(s)15.3(o)0(r)-192.2(c)0(h)19.7(i)0(p)20.7(s)]TJ
-5.7943 -1.1116 TD
[(using)-454.7(amino)-457.3(coupling)-453.7(chemistry)-451.2(according)-457(to)-451.6(the)-453.8(method)]TJ
0 -1.1174 TD
[(provid)-6.7(ed)-366.2(by)-370.8(the)-373.2(manufacturer.)-373.9(The)-368.5(MAP-tagged)-372.2(ATRXepi)]TJ
0 -1.1116 TD
[(pr)26.6(ot)26.5(e)15(i)0(n)26.5(s)-231.3(w)15.3(e)0(r)25.5(e)-232.6(d)16.2(i)16.1(lu)26.5(te)25.3(d)-231.5(i)16.1(n)-473.4(P)16.3(BS)25.7(T)-232.7(\()16.2(ph)26.6(o)16.2(s)0(p)26.8(h)16.2(at)25.3(e-)25.5(b)16.2(u)0(f)26.6(f)0(e)25.5(r)16.2(ed)-216.5(sa)25.6(l)16(i)0(n)26.5(e)0(,)-218.8(p)16.2(H)]TJ
T*
[(7.)24.3(4)16.2(,)-187.7(c)15(o)16.2(nt)26.5(a)15(i)0(n)26.5(i)16.1(ng)-175(0)16.2(.)13.9(0)16.2(0)0(5)26.6(%)-186.5(T)15(w)15.3(ee)24.3(n)-185.4(2)16.2(0\))-169.2(an)25.4(d)-185.4(i)16(nj)26.5(e)15(c)0(t)25.3(e)15(d)-185.4(a)0(t)-170.5(a)-186.6(o)26.8(w)-186.3(r)16.2(at)25.3(e)-186.6(o)16.2(f)]TJ
T*
(30)Tj
/F10 1 Tf
1.1404 0 TD
(m)Tj
/F3 1 Tf
.5414 0 TD
[(L)15(/)16.1(m)15(i)0(n)26.5(.)-279.9(Th)25.4(e)-284.5(A)15.3(T)15(R)0(X)24.7(e)15(pi)-267.3(wa)24.6(s)-283.1(d)16.2(i)16.1(l)0(u)26.5(t)16.1(ed)-268.3(in)-267.3(PB)25.7(ST)-268.2(\(1)26.6(2)16.2(.)13.9(5,)-269.5(2)16.2(5)0(,)-269.5(5)16.2(0,)]TJ
-1.6818 -1.1174 TD
[(10)26.6(0,)-402(a)15(n)16.2(d)-415.8(2)16.2(00)-140.4(n)16.2(M)0(\))-400.5(a)15(nd)-399.6(pa)25.4(s)16.4(s)0(e)25.6(d)-415.8(o)16.2(v)16.2(e)0(r)-400.7(t)0(h)26.5(e)-411.2(bi)26.5(os)26.8(e)15(n)0(s)26.8(o)0(r)-399.6(c)15(hi)26.5(p.)-402(T)15(h)16.2(e)]TJ
0 -1.1116 TD
[(bi)26.5(nd)26.6(i)16.1(n)0(g)-290.2(w)0(a)24.6(s)-306.2(m)15(o)16.2(ni)26.5(to)26.5(r)16.2(e)0(d)-291.3(f)0(o)26.6(r)-306.3(6)16.2(0)-306.4(s)16.4(e)0(c)24.3(o)16.2(nd)26.6(s,)-292.3(f)16.2(o)0(l)26.5(l)16(ow)25.7(e)15(d)-306.4(b)16.2(y)-306.4(d)16.2(i)0(s)26.7(s)0(o)26.8(c)15(ia)25.3(-)]TJ
T*
[(ti)26.3(on)-290.2(i)16.1(n)-306.4(r)16.2(u)0(n)26.6(n)16.2(in)26.5(g)-306.4(b)16.2(u)16.2(f)0(f)26.7(e)15(r)-306.3(f)16.2(or)-290.2(12)26.6(0)-300.6(s)0(e)25.6(c)15(on)26.6(ds)-290(u)16.2(s)0(i)26.7(n)0(g)-290.2(t)16(he)-291.4(Si)26.6(n)16.2(g)0(l)26.5(e)15(-c)25.5(y)16.2(c)0(l)25.3(e)]TJ
0 -1.1174 TD
[(ki)26.5(ne)25.4(t)16(i)0(c)25.3(s)-219.8(m)15(e)0(t)25.3(h)0(o)26.6(d)16.2(.)-222.3(B)15.2(in)26.5(d)16.2(i)0(n)26.5(g)-220(cu)25.4(rv)26.6(e)15(s)-219.8(we)24.6(re)-210.7(a)15(n)16.2(al)25.3(y)16.2(z)0(e)24.3(d)-220(us)26.8(in)26.5(g)-220(B)15.2(IA)25.8(c)15(o)0(r)26.6(e)]TJ
0 -1.1116 TD
[(X1)25.7(00)-359.3(E)15(v)0(a)25.4(l)0(u)26.5(a)15(ti)26.3(o)16.2(n)-375.5(S)16.3(o)0(f)26.6(t)0(w)25.6(a)15(re)-360.4(\(G)25.8(E)-376.7(H)15.3(e)15(a)0(l)25.3(t)16(hc)25.4(a)15(r)0(e)25.5(,)-372(Pi)26.6(sc)25.6(a)15(t)0(a)25.3(w)15.3(ay)25.4(,)-372.1(N)0(J)25.9(\))]TJ
T*
[(wi)25.6(th)-255.8(c)15(u)16.2(rv)26.6(e)-267.2()16.3(tt)26.3(in)26.5(g)-266(u)16.2(si)26.7(n)16.2(g)-266(a)-267.2(1)0(:)26.5(1)-266(b)16.2(i)0(n)26.5(d)0(i)26.5(n)16.2(g)-266(m)15(od)26.6(e)15(l)0(.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 478.5447 Tm
[(Western)-336(blot)-338.1(analyses)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 462.1038 Tm
-.0156 Tc
[(Ce)-15.6(l)9.7(l)-333.6(lys)-15.6(a)10(tes)-333.3(\()-15.6(1)11(0)]TJ
/F10 1 Tf
6.3645 0 TD
0 Tc
(m)Tj
/F3 1 Tf
.5357 0 TD
-.0159 Tc
[(g\))-333.7(w)-15.9(e)8.7(re)-334.9(bo)-15.9(i)10.6(l).1(ed)-333.8(in)-333.8(s)-15.9(o)10.9(diu)-15.9(m)-318.8(d)-15.9(o)10.7(decy)-15.9(l)-323.5(s).5(ul)-15.9(f)10.6(a)-.9(te)]TJ
-7.9138 -1.1174 TD
-.0157 Tc
[(\()-15.7(S)11.1(DS\))-241.4(s).7(ampl)-15.7(e)-226.5(buff)-15.7(e)9.8(r)-235.6(\(Nac)-15.7(a)8.6(l).3(ai)-235.8(Te)-15.7(s)9.9(que)-15.7(,)-223(I).5(n)-15.7(c)9.7(.\).)-238(T)-.7(he)-236.8(pro)-15.7(t)10.8(e)-.7(ins)-241.2(w)-.4(ere)]TJ
0 -1.1116 TD
[(e)-15.6(l)9.7(ect)-15.6(r)10.9(opho)-15.6(r)11(e)-.6(se)-15.6(d)-249.3(o)-15.6(n)-248.2(5)-15.6(%)9.9(20%)-265.4(po)-15.6(l)10.9(y).6(acr)-15.6(y)11(l).5(ami)-15.6(d)10.9(e)-259.8(g)-15.6(e)9.8(ls)-264.2(\(Wak)-15.6(o)-248.2(P)-15.6(u)11.2(r).6(e)]TJ
T*
[(C)-15.7(h)9.9(emic)-15.7(a)8.6(l)-293.4(Indu)-15.7(s)11.1(t).3(ri)-15.7(e)9.6(s)-293.1(Ltd.\))-293.2(a)-.7(n)-15.7(d)-277.1(t)-15.7(r)10.8(ans)-15.7(f)11.1(err)-15.7(e)9.8(d)-293.3(onto)-293.3(a)-294.4(PV)-15.7(D)9.2(F)-293.1(mem-)]TJ
T*
0 Tc
[(br)26.6(a)15(n)16.2(e)-347.8(\()0(E)25.4(M)15.3(D)-347.5(M)15.3(il)26.3(l)16(i)16.1(p)16.2(o)0(r)26.6(e)-347.8(C)15.2(o)16.2(rp)26.6(.)13.9(\))16.2(.)-349(A)15.3(f)16.2(t)16.1(er)-337.4(b)16.2(l)16.1(o)16.2(c)0(k)25.4(i)16.1(n)16.2(g)-346.7(wi)25.6(t)16(h)-346.7(4%)-337.3(s)16.4(k)16.2(i)16.1(m)]TJ
0 -1.1174 TD
-.0156 Tc
[(m)-15.6(i)9.6(lk)-258.6(\(Na)-15.6(c)8.7(alai)-258.7(T)-15.6(e)8.6(squ)-15.6(e)9.8(,)-255.2(I)-15.6(n)11(c)-.6(.\),)-260.9(t).4(he)-259.8(memb)-15.6(r)11(a)-.6(ne)-259.8(wa)-15.6(s)-249.2().7(rst)-258.7(i)-15.6(n)10.9(c)-.6(ub)-15.6(a)9.8(t).4(ed)]TJ
0 -1.1116 TD
0 Tc
[(wi)25.6(t)16.1(h)-168.1(1)]TJ
/F10 1 Tf
2.5631 0 TD
(m)Tj
/F3 1 Tf
.5357 0 TD
-.0157 Tc
[(g/mL)-190.8(PMab-)-15.7(1)10.9(,)-186.2(a)-.7(nti)-15.7(-)]TJ
/F10 1 Tf
7.6201 0 TD
0 Tc
(b)Tj
/F3 1 Tf
.5357 0 TD
-.0157 Tc
[(-)-15.7(a)9.8(ctin)-189.6(\(AC-)-15.7(1)10.9(5).5(;)-184(S)-15.7(i)10.9(g).5(ma-)-15.7(A)10.1(l).4(dr)-15.7(i)10.8(c)-.7(h,)-186.2(St)-15.7(.)]TJ
-11.2545 -1.1116 TD
0 Tc
[(Lo)25.4(u)16.2(i)16.1(s,)-459.3(M)15.3(O)0(\))25.8(,)-470(an)25.4(t)16.1(i)16(-I)26.7(D)15.3(H)15.3(1)-473.4(\()16.2(R)0(c)24.4(M)15.3(a)15(b)16.2(-1)26.6(\))16.2(,)]TJ
7.3823 0 0 6.5614 456.4345 389.7637 Tm
-.0172 Tc
[(\(21)6.7(\))]TJ
9.843 0 0 9.843 472.9889 385.3983 Tm
-.0156 Tc
[(or)-489(an)-489(an)-15.6(t)10.9(i).4(-R)-15.6(A)9.1(P)-488.8(tag)]TJ
-16.2481 -1.1174 TD
0 Tc
[(\(P)26.8(M)15.3(a)15(b-)26.6(2)16.2(\))16.2(,)-210.8(a)15(n)16.2(d)-208.4(t)16.1(h)16.2(e)0(n)-193.4(w)15.3(it)26.3(h)-208.4(1)16.2(:)16.1(1)16.2(00)26.6(0)-208.4(d)16.2(i)16(l)16.1(ut)26.5(e)15(d)-208.4(p)16.2(e)15(r)16.2(o)0(x)26.6(i)16.1(d)16.2(a)15(se)25.6(-)16.2(c)15(on)26.6(j)16(u)16.2(ga)25.4(t)16(e)15(d)]TJ
0 -1.1116 TD
.0186 Tc
[(an)6(ti)5.9(-r)6(at)-339.7(or)-345.3(m)4.8(o)6(us)6.2(e)-346.5(I)6(gG)-346.2(\()6(D)-.6(a)4.8(k)6(o;)-345.5(A)5.1(g)6(il)5.9(en)6(t)-345.5(T)4.8(ec)4.8(h)6(n).2(o)6(l).1(o)6(g).2(i)5.8(e)4.8(s,)-341.9(In)6(c.,)]TJ
T*
.0145 Tc
[(G)-4.7(l)1.8(ostrup,)-415.1(D)-4.7(enm)-5.1(a).7(rk\),)-420.9(followe)-5(d)-412.8(b)-3.9(y)-412.8(d)-3.9(evelopm)-5.1(e).7(nt)-418.7(in)-418.6(Pierce)]TJ
0 -1.1174 TD
.0129 Tc
[(W)-7.7(e)-.9(s)-5.3(t).2(er)-5.4(n)-235.9(B)-.7(l)-5.6(o).3(t)-5.6(tin)-5.5(g)-235.9(S).5(u)-5.5(b).3(s)-5.3(t).2(r)-5.4(a)-.9(t)-5.6(e)-237(Plu)-5.5(s)-235.7(\()-5.4(T)-.9(he)-6.6(rmo)-235.9(F)-5.3(is)-5.3(he)-6.6(r)-235.8(S).5(c)-6.6(i).2(en)-5.5(ti)-5.6(c)-6.6(,)]TJ
0 -1.1116 TD
[(I)-5.3(n).4(c)-6.5(.)-1.9(\))-224.2(u).4(s)-5.2(i).3(ng)-224.2(a)-225.4(S)-5.2(aya)-6.5(c)-.8(a)-225.4(I).4(m)-6.6(a)-.8(ge)-6.5(r)-224.2(\().4(D)-6.2(R)-.6(C)-225.3(C)-.6(o)-5.4(.)-220.8(Lt)-5.5(d.,)-226.6(T)-.8(ok)-5.4(yo)-5.4(,)-220.8(J)-5.2(apa)-6.5(n).4(\))-5.3(.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 308.6361 Tm
0 Tc
[(Flow)-338.7(cytometry)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 292.1952 Tm
[(The)-207.3(mouse)-203.5(anti-human)-210.6(podoplanin)-202.5(mAb)-207(LpMab-17)-207.9(\(IgG)]TJ
7.3823 0 0 6.5614 546.0093 290.5511 Tm
(1)Tj
9.843 0 0 9.843 549.751 292.1952 Tm
(,)Tj
/F10 1 Tf
-24.0468 -1.1116 TD
(k)Tj
/F3 1 Tf
.5529 0 TD
[(\))-243(was)-241.3(described)-243.7(previously.)]TJ
7.3823 0 0 6.5614 426.0471 285.6188 Tm
(\(22\))Tj
9.843 0 0 9.843 440.7306 281.2535 Tm
[(Cell)-243(lines)-242.9(were)-238(harvested)-243.7(by)]TJ
-12.9709 -1.1116 TD
[(brief)-605.7(exposure)-604.2(to)-607.1(0.25%)-602.2(Trypsin/1)-166.7(mM)-603.3(EDTA)-607.5(\(Nacalai)]TJ
0 -1.1174 TD
[(Tesque,)-232.2(Inc.\).)-225.5(After)-226.5(washing)-228.6(with)-224.4(PBS,)-226.3(the)-223.5(cells)-226.8(were)-226.4(treated)]TJ
0 -1.1116 TD
[(with)-558.5(PMab-1)-555.7(\(1)]TJ
/F10 1 Tf
7.1132 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(g/mL\))-555.2(or)-555.1(LpMab-17)-559.2(\(1)]TJ
/F10 1 Tf
10.2868 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(g/mL\))-555.2(for)-556.2(30)]TJ
-18.5059 -1.1116 TD
[(minutes)-361.5(at)-360.6(4)]TJ
/F8 1 Tf
5.1204 0 TD
()Tj
/F3 1 Tf
.3974 0 TD
[(C,)-356.9(followed)-362.4(by)-353.5(treatment)-360.6(with)-356.9(1:1000)-358.1(diluted)]TJ
-5.5178 -1.1174 TD
[(Oregon)-192.9(Green)-193(488)-187.6(goat)-190(anti-rat)-190(or)-192.2(mouse)-186.3(IgG)-194.2(\(Thermo)-189.8(Fisher)]TJ
0 -1.1116 TD
[(Scientic,)-431.6(Inc.\).)-427.1(Fluorescence)-427.7(data)-427.3(were)-428(collected)-423.9(using)-425.9(a)]TJ
T*
[(Cell)-283.3(Analyzer)-284(EC800)-284.2(\(Sony)-281.8(Corp.,)-283(Tokyo,)-280.8(Japan\).)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 182.6078 Tm
[(Protein)-336.3(expression)-335.9(and)-335.7(purication)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 166.1669 Tm
[(PMab-1)-486.5(was)-489(coupled)-488.2(to)-486.1(CNBr-activated)-495.6(Sepharose)-485.3(4B)]TJ
-1.0137 -1.1116 TD
[(\(GE)-356.7(Healthcare\))-364.6(according)-353.3(to)-359.4(the)-350.2(protocol)-355.8(provided)-355.7(by)-359.3(the)]TJ
0 -1.1174 TD
[(manufactur)-10(er,)-352.3(routinely)-357(yielding)-361.7(coupling)-355.8(levels)-360.3(of)]TJ
/F10 1 Tf
21.3455 0 TD
(*)Tj
/F3 1 Tf
1.0022 0 TD
.1681 Tc
[(2m)168.1(g)]TJ
-22.3477 -1.1116 TD
0 Tc
[(IgG/mL)-453.5(gel.)-445.8(Recombinant)-455.9(ATRXepi)-450.7(\(PA-ATRXepi-RAP-)]TJ
T*
[(MAP\))-443.7(was)-442.9(expressed)-445(in)-440.1(transformed)]TJ
/F7 1 Tf
15.8162 0 TD
[(E.)-443.5(coli)]TJ
/F3 1 Tf
3.2542 0 TD
[(BL21)-444.4(\(DE3\))]TJ
-19.0704 -1.1174 TD
[(cultured)-201.4(for)]TJ
/F10 1 Tf
4.8554 0 TD
(*)Tj
/F3 1 Tf
1.0022 0 TD
[(3)-196.9(hours)-195.4(at)-199.3(37)]TJ
/F8 1 Tf
5.034 0 TD
()Tj
/F3 1 Tf
.3974 0 TD
[(C)-198(i)0(n)-198.2(the)-194.7(presence)-203.3(of)-198(1)-168.1(m)0(M)-194.4(IPTG,)]TJ
-11.289 -1.1116 TD
[(and)-217.6(the)-217.7(bacterial)-222.3(pellet)-216.7(from)-212.9(a)-215.4(100)-170.3(mL)-217.7(culture)-217.6(was)-212.5(suspended)]TJ
T*
[(in)-180.9(10)-169.2(mL)-183.2(of)-180.7(Tris-buffered)-184.7(saline)-181.8(\(TBS;)-186.2(pH)-175.9(7.5\).)-182.9(Bacteria)-187.5(were)]TJ
0 -1.1174 TD
[(lysed)-237(by)-232.6(sonication,)-235.8(and)-234.9(the)-235(soluble)-233.6(fraction)-236(was)-229.8(obtained)-236(by)]TJ
0 -1.1116 TD
[(centrifugat)-10.2(ion)-314.5(at)-314.5(15,000)]TJ
/F7 1 Tf
10.068 0 TD
(g)Tj
/F3 1 Tf
.8121 0 TD
[(for)-320(15)-313.2(minutes.)-318.9(The)-316.7(lysates)-320.9(were)]TJ
-10.8801 -1.1116 TD
[(passed)-262(through)-261.2(a)-255.7(0.45)]TJ
/F10 1 Tf
8.9218 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(m)-255.8(lter)-260.2(to)-261.5(remove)-257.8(any)-263.6(trace)-256.8(amounts)]TJ
/F5 1 Tf
8.9663 0 0 8.9663 62.022 749.6502 Tm
(294)Tj
48.6548 0 TD
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