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. 2018 Feb 14;8:3048. doi: 10.1038/s41598-018-21322-z

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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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108-B6 and 4A68 recognize PGRMC1 on the surface of NT-2/D1 and H9 hPSCs. (a) 108-B6 and 4A68 recognize approximately 25 kDa cell surface proteins. NT-2/D1 cells were biotinylated, and cell lysates were subjected to immunoprecipitation with 108-B6 or 4A68 and visualized with SA-HRP. Immunoprecipitated proteins (approximately 25 kDa) are indicated by an arrowhead. Con represents immunoprecipitation with Protein G-Sepharose beads alone. (b) 108-B6 and 4A68 recognize the same PGRMC1 protein. NT-2/D1 cell lysates were immunoprecipitated with 108-B6 or 4A68. The immunoprecipitated proteins were analyzed by Western blot with 108-B6 or α-PGRMC1 followed by incubation with HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG, respectively. The monomer (M) and trimer (T) forms of PGRMC1 were detected and indicated by arrowheads (top panel). HC represents immunoglobulin heavy chain. Full-length blots are presented in Supplementary Figure 8. (c) 108-B6 and α-PGRMC1 recognize the same PGRMC1 protein in hPSCs. H9 hPSC lysates were immunoprecipitated with 108-B6 and α-PGRMC1, and the immunoprecipitated proteins were detected by Western blot with α-PGRMC1 or 108-B6 followed by incubation with HRP-conjugated rabbit IgG or HRP-conjugated mouse IgG, respectively. Full-length blots are presented in Supplementary Figure 8. (d) Overexpression and detection of PGRMC1 with 108-B6, 4A68 or α-PGRMC1. HEK293T cells were transfected with pcDNA3.1+ (vector), pCMV-SPORT6-HSPA8 (irrelevant plasmid) or pCMV-SPORT6-PGRMC1 (PGRMC1). The cell lysates were subjected to Western blot with 108-B6, 4A68, or α-PGRMC1. The monomer (M) and trimer (T) forms of PGRMC1 were detected and indicated by arrowheads (top panel). β-actin was used as internal protein control and loading control. Full-length blots are presented in Supplementary Figure 8. In (a–d), images are representative of at least two independent experiments. (e) Flow cytometric analysis of H9 hPSCs with 108-B6, 4A68, and α-PGRMC1.