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. 2018 Jul 17;8:10780. doi: 10.1038/s41598-018-29236-6

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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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Generation of HEK293 cells overexpressing secreted and active forms of LOX and BMP1 proteins. Induction of LOX (A) and BMP1 (B) proteins in HEK293 cells upon incubation with the tetracycline analog, doxycycline (Dox), at 10 μM as assessed by western blotting using total cell extracts or Amicon-concentrated aliquots of the cell supernatants. (C) Combination of cell supernatants containing LOX and BMP1 proteins gives rise to the proteolytic activation of LOX as assessed by western blotting. The blot shown correspond to a representative experiment performed twice with two independent preparations. (D) LOX-immunoreactive bands from results shown in panel (C) were quantified and expressed as percentage of total: 50 KDa precursor (closed circle), 30 KDa active form (open circle), and 25 KDa unknown band (open squares). (E) LOX enzymatic activity as measured using Amplex red assay in cell supernatants from uninduced cells (Basal, white bar) or induced with Dox and incubated with BMP1 for 60 min (LOX + BMP1, closed bars). Values are represented as arbitrary fluorescent units (mean ± SEM, n = 6; *P < 0.01).