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. 2020 Nov 4;11:5570. doi: 10.1038/s41467-020-19373-w

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© The Author(s) 2020

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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Fig. 6 — a Western blot analysis in H1581 cells treated with siControl or siRSPO2 as indicated and transfected with or without BMPR1A-HA DNA. Transferrin receptor (TfR), a loading control. Ratio, relative levels of BMPR1A-HA normalized to TfR. Data shows representative result from 3 independent experiments with similar conclusion. b Western blot analysis of Bmpr1a in Xenopus laevis (St. 15) neurulae injected animally at 2-cell to 4-cell stages as indicated. GFP mRNA was injected as an injection control. Ratio, relative levels of Bmpr1a normalized to GFP. Data shows representative result from 3 independent experiments with similar conclusion. c Scheme for immunofluorescence microscopy (IF) in Xenopus laevis animal cap (AC) explants. Embryos were injected animally at 4-cell stage with bmpr1a-EYFP and memb-RFP mRNA along with bmp4 and rspo2 wild-type or mutant mRNA. AC explants were dissected at St.9 for IF. Membrane (memb)-RFP was used as a control comparing relative change of Bmpr1a-EYFP signal at cell surface. d IF for Bmpr1a (green) and cell membrane (red) in AC explants injected as indicated, with a representative cell (top) and magnification (inset). Scale bar, 20 μm. e Quantification of d. n = the number of areas analyzed and data are displayed as mean ± SD. ns, not significant; ****P < 0.0001 from two-tailed unpaired t-test. f Scheme for IF in Xenopus laevis ventrolateral marginal zone explants (VLMZ). Embryos were ventrally injected at 4-cell stage with bmpr1a-EYFP and memb-RFP mRNA with Mo. VLMZs were dissected at stage 11.5 for IF. g IF for Bmpr1a (green) and cell membrane (memb-RFP, red) in VLMZ injected with mRNA and Mo as indicated. Scale bar, 20 μm. h Quantification of g. n = the number of areas analyzed and data are displayed as mean ± SD. **P < 0.01, ***P < 0.001 from two-tailed unpaired t-test. i Western blot analysis of Bmpr1a in Xenopus laevis neurulae (St. 18) injected radially at 4-cell stage as indicated. Gapdh, a loading control. Data shows representative result from 2 independent experiments with similar conclusion.