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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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Panx3 is phosphorylated. (A,a) A phosphorylation band was revealed from pEF1/Panx3 cell lysates by Pro-Q Diamond phosphoprotein gel staining. Control (pEF1) cells and C2C12 cells transiently transfected with Panx3 (pEF1/Panx3) were cultured for 2 days in DMEM containing 10% FBS. Immunoprecipitated cell lysates were loaded onto NuPAGE Bis-Tris gels. After electrophoresis, the gels were fixed and stained with Pro-Q stain. Lysates were incubated with CIP (ALP:40 or 80 U) for 45 min at 30 °C before loading the gel. Lysates were analyzed by western blotting with a α-tubulin as a control, to confirm loading of the same amount of protein (bottom). (b) Quantification of the ratios of Pro-Q stained band/V5. (B,a) Immunoprecipitation assays with cell lysates from pEF1/Panx3 transiently transfected cells showed a phosphorylation band of serine and threonine which was reduced by CIP. The Panx3 protein overexpressed in C2C12 cells was precipitated with anti-V5 antibody and analyzed by immunoblotting with Phospho-Serine and Threonine (P-Ser Thr) antibody. Precipitated lysates were treated with CIP (80U) before loading. (b) Quantification of the ratios of P-Ser Thr/V5. *P < 0.01. Error bars represent the mean ± SD; n = 3. Pro-Q stain and Western blot were perfomed by at least three independent experiments for each experimental group. Full blot is shown in the Supplemental Information (Full Original Blots-I).