Figure 3: EB1 binds to Ska1 through its C terminus.

(a) Regions of various deletion constructs of EB1 (C and N terminus) and Ska1 are shown. (b) In vitro GST pull-down with purified human full length EB1–GST and purified human His-tagged Ska1. Purified GST protein was used as control. (c) Far-western blot analysis with immobilized EB1, EB1n and EB1c followed by incubation with purified Ska1. Immobilized BSA and tubulin were used as negative and positive control, respectively. Reverse Far-western with immobilized Ska1 followed by incubation with purified His-EB1 or His-EB1c is shown. Rabbit polyclonal EB1 antibody targeted against the C-terminal region of EB1 was used. It could recognize both the full length EB1and EB1 C terminus. (d) The western blot shows in vitro GST pull-down using purified EB1c–GST and purified Ska1. (e) Lysates of synchronized mitotic HeLa cells was incubated with purified EB1c–GST followed by GST pull down using Sepharose beads and analysed by western blot. (f) Purified Ska1 and EB1 or the mixture EB1 and Ska1 was loaded on Superose 12 column and the fractions (200 μl) were analysed by Western blot. Molecular weights of Ska1 and EB1 monomers are 30 and 32 kDa, respectively. (g) Western blot image shows GST pull down in vitro using purified EB1-GST and purified Ska complex (mixture of Ska1, 2 and Ska3 in 1:1:1 ratio).