Figure 1. Aurora B is a CaM binding protein. (A) CaM-sepharose pull-down (PD) assays showing no effects of exogenous calcium on binding between CaM and endogenous Aurora B. (B) CaM-sepharose PD assays showing binding of in vitro synthesized full-length (FL) or various Aurora B deletion mutants to CaM. (C) CaM-sepharose PD assays showing binding of in vitro synthesized FL or point mutants of Aurora B. (D) Murine lung epithelial (MLE) cells were transfected with FL or a V5-Aurora B variant harboring a point mutation within the IQ motif followed by co-immunoprecipitation of endogenous FBXL2 and V5-immunoblotting. HC = heavy chain. (E) Purified GST-FBXL2 was incubated with in vitro synthesized V5- tagged FL or a V5-Aurora B IQ mutant, followed by GST pull-down. PD products were resolved on SDS-PAGE followed by V5 immunoblotting. (F) In vitro ubiquitination assays. Purified SCF complexes were incubated with Aurora B point mutants and the full complement of ubiquitination reaction components. (G) Aurora B protein half-life determination after expression of V5-Aurora B point mutants (n = 2 experiments).