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. 2016 Jun 22;5(7):962–969. doi: 10.1242/bio.018572

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© 2016. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 6. — Pull-down analysis using reducing SDS-PAGE. (A) As expected, MICU1-NΔ54 interacts with MICU2-NΔ66 and MICU2-NΔ84 which contain Cys413. (B) In the absence of Cys413, MICU1-NΔ96 still interacts with MICU2-NΔ66-CΔ36 and MICU2-NΔ84-CΔ36. (C) The EF-hands of MICU2 are not involved in its interaction with MICU1. (D,E) In addition to the disulfide bond, salt bridge formed by Arg221 in MICU1 and Asp330 in MICU2 contributes to the MICU1-MICU2 interaction. (F) Western blot analysis of co-immunoprecipitation of MICU1-Δ96-Myc with MICU2-NΔ84-Flag and MICU2-NΔ84-CΔ28-Flag reveals the interaction both in the presence and absence of Cys413, consistent with the in vitro pull-down results (A,B). The white vertical line refers to the fact that MW markers were loaded on the same SDS-PAGE, but not in the adjacent lanes.