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. 2017 May 1;130(9):1637–1651. doi: 10.1242/jcs.198267

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© 2017. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — Interaction between KHC and SKIP requires both W-acidic motifs. (A) Fluorescence polarization binding assay using a TAMRA-conjugated peptide from SKIP containing both W-acidic motifs. KHC(866-917) was prepared by cleavage of purified GST–KHC(866-917) and removal of free GST or uncleaved protein by incubation with glutathione beads. KHC(866-917) is added at increasing concentration. Error bars show s.e.m. from three replicates and curve shows fit of data to a one-site specific binding model with hill slope [EQ Y=B_max×X^h/(K_d^h+X^h) where B_max=180.7, K_d=7.878] (h)=4.703. R²=0.98. (B) Western blot (IB) showing GST pulldown experiment from extracts of 293T cells expressing wild-type (WT) GFP–SKIP(1-310) or WD/AA, WE/AA or WDWE/AAAA mutants. Binding is reduced by disruption of either motif. Data are representative of three independent experiments. (C) Western blot showing GST pulldown experiment using purified His₆–SKIP(1-310) produced in E. coli. Binding is reduced by disruption of either W-acidic motif. Data are representative of three independent experiments.