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. 2019 Dec 10;9:18759. doi: 10.1038/s41598-019-55371-9

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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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Generation of P-Panx3 specific antibody and its localization in the tibial growth plate of newborn mice. (A) Specificity evaluation of phosphorylated Panx3 at Ser68 antibody (P-Panx3) against antigen-peptide (P-peptide) or control (Native peptide) by ELISA. ELISA was performed in dishes coated with serial dilutions of P-Panx3 peptide. Error bars represent the mean ± SD; n = 6. (B) Western blots, using P-Panx3 antibody and lysates from C2C12 cells stably transfected with pEF1/Panx3 and Ser68Ala. Protein was isolated after 1 day of culture in DMEM. For dephosphorylation, protein from pEF1/Panx3 cells was treated CIP (80U) before loading. V5 antibody was used for detection of Panx3 expression. (C) Western blots using P-Panx3 and Panx3 antibodies and tissue lysates isolated from brain, skin and bone + cartilage of newborn mice. (D) Immunohistochemistry of newborn mouse growth plates. Image observed under light microscopy (black & white, left vertical lane), P-Panx3 (green, upper and middle lane), Panx3 (green, bottom lane), Ocn (red), Hoechst nuclear staining (blue) and merged image (right vertical lane) The middle lane is staining images of P-Panx3 in the presence of its antigen peptide (P-peptide). Western blot was perfomed by at least three independent experiments. Full blot is shown in the Supplemental Information (Full Original Blots-III).