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. 2018 Feb 21;8:3420. doi: 10.1038/s41598-018-21571-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Limited proteolysis pattern of CaM in the presence and in the absence of NPs. (a) Proteolysis reactions were performed at 25 °C by 30–120 min incubation with saturating (2 mM) EGTA or Ca²⁺, using a CaM:TPCK-trypsin ratio equal to 1:60. Lane M refers to the protein marker. (b) Proteolysis experiments in the presence of NPs were performed by 30 min incubation with saturating (240 µM) EGTA or Ca²⁺, using a CaM:TPCK-trypsin ratio equal to 1:60. Lanes 3 and 5 refer to the digested apo (lane 3) and Ca²⁺-bound (lane 5) CaM. Lanes 8 and 10 refer to the digested CaM previously incubated with NPs (lane 8) and after the addition of EGTA (lane 10). Undigested CaM was loaded in the same conditions in lanes 2, 4, 7 and 9. The similar effect exerted by either free Ca²⁺ or NPs and excess EGTA on the proteolytic patterns is highlighted by downwards and upwards arrows, respectively. The figure results from two separate gels, which have been reported in full-length in Supplementary Figure S1.