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. 2018 Aug 15;8:12236. doi: 10.1038/s41598-018-30362-4

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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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Studies on the intracellular behavior of CRELD2 protein in living Neuro2a cells using a NanoLuc complementary reporter assay. (A) Schematic structures of NanoBiT-tagged constructs used in this study. (B) After transfection of the indicated CRELD2 gene into the cloned CRELD2-deficient cells, expression of the indicated protein was determined as described in the Materials and methods section. (C) Twenty-hour hours after transfection of the indicated genes into the cloned CRELD2 deficient cells, culture medium was replaced with OPTI-MEM and cells were cultured for additional 6 h. After incubation, culture medium was collected for measurement of extracellular NanoBiT activity (open bars). For measurement of intracellular NanoBiT activity, culture medium was replaced with fresh OPTI-MEM, and the diluted substrate was directly added to each well to measure the NanoBiT activity (filled bars). (D) Thirty hours after transfection of SP-SmBiT-CRELD2 together with the indicated gene, culture medium was replaced with fresh OPTI-MEM and the diluted substrate was directly added to each well to measure the intracellular NanoBiT activity in living cells. Each value represents the mean ± SEM from 3 independent cultures.