Journal: Open Biology
Article Title: The Ndc80 complex targets Bod1 to human mitotic kinetochores
Figure Lengend Snippet: The Ndc80 complex interacts with Bod1 in mitotic HeLa cell extracts and in vitro . ( a ) Graphic representation of the full-length Ndc80 complex. Domains of interest, including the microtubule and centromere binding regions of the complex, are labelled. ( b ) Co-precipitation of Ndc80 complex components Ndc80, Nuf2 and Spc24 from mitotic HeLa cell extracts with purified Bod1-GST. ( c ) Representation of the recombinant Ndc80 Bonsai complex (as described in [ 38 ]). Ndc80–Spc25 and GST–Nuf2–Spc24 are expressed as fusion proteins. Both fusion constructs are co-expressed in E. coli from a dual-expression vector. ( d ) Of note, 150 pmol recombinant Ndc80 Bonsai, consisting of dimers of one Ndc80–Spc25 fusion protein with one GST–Nuf2–Spc24 fusion protein, was immobilized on Sepharose beads and incubated with 1 nmol Bod1-MBP or MBP. Binding was allowed for 1 h. Proteins were resolved by SDS-PAGE and immunoblotted using simultaneous detection of the MBP (red) epitope tag on Bod1 and the GST (green) epitope tag on Nuf2–Spc24. ( e ) Amount of bound protein in ( d ) was quantified relative to the input. Two asterisks indicate level of significance ( p
Article Snippet: Primary antibodies included mouse anti-B56α (1 : 500, Abcam), mouse anti-B56δ (1 : 500, Abcam), mouse anti-Ndc80 (1 : 1000, Abcam [9G3]), mouse anti-Nuf2 (1 : 1000, Abcam), rabbit anti-Spc24 (1 : 1000, Abcam [EPR11548(B)]), mouse anti-MBP (1 : 20 000, NEB), goat anti-GST (1 : 5000, Abcam), mouse anti-Vinculin (1 : 10 000, Abcam [SPM227]), mouse anti-GFP (1 : 1000, Roche), rabbit anti-Bod1 (1 : 500, Abcam), polyclonal sheep anti-Bod1 (2 µg ml−1 ).
Techniques: In Vitro, Binding Assay, Purification, Recombinant, Construct, Expressing, Plasmid Preparation, Incubation, SDS Page