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Figure 4.
Figure 4 Transferability of the NZF [13]TF[14]/M[25] Ub-binding
motif. (A) Schematic alignment of the Npl4 and RanBP2 NZF
sequences. Dark blue: highly conserved NZF residues (>50%);
light blue: moderately conserved NZF residues (>20%); yellow:
residues that contact Ub; and green: residues that are both
highly conserved and contact Ub. NZF residue conservation was
defined as in Wang et al (2003). (B) Ub binding by wt (inset)
and mutant RanBP2 (L13T,V14F,A25M) NZF domains. Ub was injected
in triplicate at concentrations of 0 -1500  M
over GST-RanBP2 NZF proteins captured on anti-GST surfaces. Note
that Ub binding by the wt RanBP2 NZF domain (inset) was
negligible, even at 1500 M
Ub. (C) Binding isotherms for the NZF domains of Npl4 (positive
control, black), wt RanBP2 (negative control, purple), RanBP2
(L13T,V14F) (blue), RanBP2 (L13T,V14F,A25M) (green), and RanBP2
(L13T,V14F,A24E,A25M) (red). K[d] values are given in Table II.
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