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. 2019 Dec 9;9:18635. doi: 10.1038/s41598-019-54566-4

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© The Author(s) 2019

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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Expression of bone-secreted proteins in the calvariae. (A) Western blots. A 20X concentrated BSA-free Calvarial-CM (Bone-CM) together with a 20X concentrated control BGJb medium were incubated with antibodies against bone-secreted factors as indicated. Original Western blots are shown in Supplemental Fig. S1. (B) ELISA. Protein levels of select bone-secreted factors in two independent preparations of mouse Calvarial-CM, Calvarial-CM #1 and Calvarial-CM #2, were analyzed by ELISA. m, mouse. (C) Real-time qRT-PCR. Total RNAs were prepared from PMO versus total calvariae and analyzed by qRT-PCR. mRNA levels normalized against GAPDH controls were compared between PMO versus calvariae. A higher PMO to calvariae ratio indicates that the protein is expressed by osteoblasts. Error bars represent triplicate determinations.