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. Author manuscript; available in PMC: 2015 Mar 18.
Published in final edited form as: Nat Commun. 2014 Sep 18;5:4903. doi: 10.1038/ncomms5903

Figure 3. WT1 has higher affinity for the active closed MAD2 conformer.

Figure 3

(a) GST-interaction assays were carried out with WT1 (residues 245–297) and full length (FL) His-MAD2 alone and also with MAD2 pre-incubated with either 40µM of MAD2-binding peptide 1 (MBP1), or a control peptide. The pulldown complexes were resolved by SDS-PAGE and immunoblotted with anti-MAD2 antibodies. (b) In vitro pulldown assays were performed with either Flag-M2 magnetic beads alone or full length Flag-tagged WT1 protein in the presence of Full length His-MAD2 (pre-incubated with MBP1) or the MAD2 delC mutant. The interaction was analyzed by immunoblotting with anti-WT1 and anti-MAD2 antibodies. (c) GST-interaction assays were carried out with His-MAD2, MAD2 L13A and MAD2-C-terminal deletion mutant (Del-C). Immunoblotting was performed using anti-MAD2 and anti- GST antibodies. (d) GST-interaction assays were carried out with His-MAD2 + MBP1, MAD2-Del-C, and MAD2 dimerization mutant, R133A. Immunoblotting was performed using anti-MAD2 and anti-GST antibodies. (e) Pull down assays were performed with full length Flag-WT1 protein and the different MAD2 point mutant derivatives. Immunoblotting was performed using anti-MAD2 and anti-WT1 antibodies. Input is 20% of the different MAD2 proteins used for interaction analysis.