Figure 6
EB1 acetylation at K220 may regulate the MT plus-end loading and tracking of DDA3.
(a) Acetylation of EB1 at K220 inhibited DDA3-EB1 interaction. GST-DDA3 was used as affinity matrix to pull down His-EB1191–268 and pre-acetylation-treated His-EB1191–268. Western blotting analyses were then performed with His antibody and anti-acK220 antibody to indicate specific interaction. (b) EB1K220Q inhibited MT plus-end tracking of DDA3 in cells. HeLa cells were co-transfected with mCherry-DDA3, EB1 shRNA and EB1-GFP or its mutant for 72 hr and then live cell images were collected. (c) Statistical analysis of MT plus-end localization of mCherry-DDA3 in b, as described in Fig. 2g. ***, P < 0.001 by t-test. (d, e) TIRF experiments, performed in vitro, indicated that EB1K220Q inhibits MT plus-end tracking of DDA3. The corresponding kymograph at the bottom shows the same MT in a period of 2 min. (f) Statistical analysis of the relative intensity of DDA3122–363 at MT plus ends in d and e, as described in Fig. 2j. **, P < 0.01 by t-test. Scale bars, 5 μm (all image panels).
