Supplementary Material
SUPPLEMENTARY FIGURE 1
Cleavage sites for different proteases on mouse and human proNGF.
The aminoacid sequences of mouse and human proNGF were aligned, and the cleavage sites for
trypsin, furin and plasmin were mapped onto the sequences (for the prediction, the software
Peptide Cutter was used - http://web.expasy.org/peptide_cutter/).
Legend:
Italics: signal sequence for proNGF secretion
Underlined: pro-peptide
Normal text: mature NGF
Green: cleavage sites for trypsin and plasmin, equal in the two sequences
Yellow: cleavage sites for trypsin and plasmin, equal in the two sequences, but with different
cleavage probability for trypsin
Magenta: cleavage sites for trypsin and plasmin, different in the two sequences
Bold, double underlined: furin cleavage site
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SUPPLEMENTARY FIGURE 2
Detailed Biacore kinetics for the binding to the anti-NGF MAB 256 R&D System
Detailed SPR binding kinetics of the neurotrophins over the anti-NGF antibody R&D MAB 253. A mouse NGF, B - mouse proNGF, C - human NGF, D - human proNGF. Concentrations used, from
top to bottom: 100, 50, 25, 6.3, 3.1, 1.6, 0.8, 0.4, 0.2, 0.1 nM.
2
SUPPLEMENTARY FIGURE 3
Detailed Biacore kinetics for the binding to the anti-NGF MAb D11
Detailed SPR binding kinetics of the neurotrophins over the anti-NGF antibody MAb D11. A mouse NGF, B - mouse proNGF, C - human NGF, D - human proNGF. Concentrations used, from
top to bottom: 100, 50, 25, 6.3, 3.1, 1.6, 0.8, 0.4, 0.2, 0.1 nM.
3
SUPPLEMENTARY FIGURE 4
Detailed Biacore kinetics for the binding to the anti-proNGF MAb Millipore clone EP1318Y
Detailed SPR binding kinetics of the neurotrophins over the anti-proNGF MAb Millipore clone
EP1318Y. A - mouse proNGF, B - human proNGF. Concentrations used, from top to bottom: 100,
50, 25, 6.3, 3.1, 1.6, 0.8, 0.4, 0.2, 0.1 nM.
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SUPPLEMENTARY TABLE 1
Summary of binding kinetics of the mouse and human NGF
MAb anti-NGF R&D
MAb anti-NGF αD11
mNGF
hNGF
ka (M-1s-1)
5.20 x 106
8.32 x 106
kd (s-1)
1.03 x 10-3
8.64 x 10-4
2
20
7
ka (M-1s-1)
1.20 x 107
4.31 x 106
kd (s-1)
2.07 x 10-15
3.63 x 10-7
2
0
0
SPR analysis: Summary of the derived kinetic binding constants of mNGF and hNGF towards the
MAbs anti-NGF MAB 256 R&D and D11 .
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SUPPLEMENTARY TABLE 2
Summary of binding kinetics of the mouse and human proNGF
MAb anti-NGF R&D
MAb anti-NGF αD11
MAb anti-proNGF
Millipore
mproNGF
hproNGF
ka (M-1s-1)
9.98 x 105
5.37 x 104
kd (s-1)
1.81 x 10-3
1.89 x 10-3
2
30
6
ka (M-1s-1)
3.57 x 106
1.20 x 106
kd (s-1)
14.60 x 10-3
3.63 x 10-7
2
13
8
ka (M-1s-1)
2.82 x 104
2.34 x 105
kd (s-1)
4.84 x 10-4
1.22 x 10-3
2
9
18
SPR analysis: Summary of the derived kinetic binding constants of mproNGF and hproNGF
towards the MAbs anti-NGF MAB 256 R&D and D11 and tha MAb anti proNGF Millipore clone
EP1318Y.
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