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. 2019 Dec 27;9:20257. doi: 10.1038/s41598-019-56208-1

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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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Bone marrow derived c-Cbl^+/− macrophages express higher levels of PD-1. (A) BMDM differentiated from c-Cbl^+/+ and c-Cbl^+/− for 7 days were subjected to FACS analysis. The cells were gated using CD11b and PD-1. Percentage of PD-1+ macrophages are shown. Fluorescence minus one (FMO) served as a control. Representative FACS plots from three independent experiments are shown. (B) Average from three independent experiments is shown. Student’s t-test were performed. Error bars = SEM. *p < 0.001. (C) Lysates of BMDMs from mice differentiated for 7 days and treated with LPS overnight. Representative immunoblots from three independent experiments. Student’s t-test with Bonferroni correction was performed. *p = 0.004 for PD-1 in c-Cbl^+/+ macrophages with and without LPS, **p = 0.001 for PD-1 between c-Cbl^+/+ and c-Cbl^+/− macrophages without LPS and ^#p = 0.02 for LPS-treated c-Cbl^+/+ and c-Cbl^+/− macrophages. Here, and in all other blots, molecular weights in kilo Dalton are denoted on the left. (D) BMDM from c-Cbl^+/+ (red line) and c-Cbl^+/− (blue line) mice differentiated for 7 days and treated with LPS overnight were examined using FACS for PDL-1. Representative MFI of PDL-1 from three independent pairs of mice is shown. (E) Average MFI of PDL-1 on BMDM obtained from both the groups (N = 3 mice/group) is shown. Student’s t-test were performed. Error bars = SEM. p = 0.3.