Extended Data Fig. 8: The interaction between EB1 and NuMA is mediated by EEY motif.
From: Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis
(a) EB1 binding of purified + TIP fragments in vitro. Recombinant + TIP proteins were purified and incubated with recombinant wild-type, AcK66- and CrK66-EB1 for pull-down assay. Their interaction was assessed by Western blotting with an anti-EB1 antibody. (b) Representative immunofluorescence images of endogenous EB1-depleted HeLa cells expressing EB1-WT-GFP and CrK66-EB1-GFP. Cells were fixed and stained with NuMA and α-tubulin antibodies. The boxed areas are magnified in the right panels to show details of the astral microtubule region. Scale bar, 10 μm. For the whole-cell images were shown as Z-stacks, while zoom-in images as single-plane images. Arrowheads indicate the cortex NuMA in whole-cell images, and arrowheads indicate EB1 dots that co-localize with cortex NuMA. (c) Endogenous EB1-depleted HeLa cells expressing EB1-WT-GFP or CrK66-EB1-GFP, AcK66-EB1-GFP with mCherry-LGN. The whole-cell and kymograph images of indicated region were shown. Arrowheads indicate EB1 near to the cortex. (d) In vitro pull-down assay. Recombinant EB1, AcK66-EB1 and CrK66-EB1 were captured on Ni-NTA resin followed by incubation with mitotic HeLa cell lysates. The binding fractions were analyzed by Western blotting and mass spectrometry. (e) Sequence alignment of NuMA homologs from different species. Two conserved SxIP motifs in NuMA-CT were annotated with blue pentastar. (f) Bacterially expressed GST-NuMA-CT was purified and used as affinity matrix to isolate His-EB1-WT and CrK66. Binding activity was analyzed with an anti-EB1 antibody and protein levels were shown by staining with CBB. (g) Recombinant GST-NuMA-CT and SxIP-motif mutants were incubated with His-EB1 for GST pull-down assay. NN#1, NN#2 and NNNN were the asparagine-substituted mutants of the SxIP motifs in e. (h) Recombinant GST-NuMA-CT and its deletion mutants were incubated with His-EB1 for GST pull-down assay. Their interaction was assessed by Western blotting with an anti-EB1 antibody and protein levels were shown by staining with CBB. Related to Fig. 5 & Supplementary Table 2.
