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. 2016 Jun 7;24(6):851–861. doi: 10.1016/j.str.2016.03.020

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© 2016 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

CD-Mediated Ca²⁺/CaM Binding in hDAPK2

(A) CaM pull-down assays of purified hDAPK2 variants monitored by SDS-PAGE Coomassie blue staining. ND, not determined. Data are normalized to the hDAPK2 S308A mutant. Error bars represent one SD from three experimental repetitions.

(B) CaM pull-down assays of hDAPK2 variants HEK293T transfected cells monitored by western blot. Data are normalized to the hDAPK2 W305D mutant. I, cell input; E, EGTA-mediated elution on Sepharose 4B-CaM; C, EGTA-mediated elution on Sepharose 4B without CaM (negative control). Relative units were calculated using the average ratio between input and elution band intensities for each mutant over three replicates. Error bars represent one SD from three experimental repetitions.

(C and D) In vitro fluorescence anisotropy assays of purified hDAPK2 variants titrated against Ca²⁺/CrAsH-CaM. Error bars represent one SD between the triplicates of a representative experimental curve. K_D values were estimated using a non-linear regression assuming one-site specific binding with the software Prism (version 5.0, GraphPad) for three experiments.