Skip to main content
. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: Exp Mol Pathol. 2009 Dec 23;88(2):225–233. doi: 10.1016/j.yexmp.2009.12.004

Fig. 2.

Fig. 2

Phosphorylation of Mad2 by Nek2. (A) Purified recombinant CBP-Mad2 protein (10 µg) was incubated with the indicated amount of Nek2 (ng) in the presence of 32P-γ-ATP. Protein phosphorylation was evaluated by SDS–PAGE and phosphorimaging. (B) In vivo phosphorylation of Mad2 by Nek2. 293T cells were transfected with the plasmids as indicated followed by metabolic labeling. Immunoprecipitation (IP) was performed using anti-FLAG antibody. Top panel: Phosphorylation was evaluated by phosphorimaging. Middle and bottom panels: Western blot analysis using anti-FLAG and anti-GFP antibody, respectively. (C) FLAG-Mad2 or its mutants were coexpressed with GFP-Nek2A in 293T cells followed by metabolic labeling. IP and phosphorimaging were performed in the same way as in (B). The Mad2 mutants are Mad2-133/134 (R133E, Q134A) and Mad2-mut3 (S170G, S178A, S195A).