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. 2021 Mar 19;12:1720. doi: 10.1038/s41467-021-21961-3

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© The Author(s) 2021

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. 4 — a Cycloheximide-chase assay for BMPRII in PAECs treated with either vehicle or ActA (20 ng/mL) (n = 3 biologically independent cells in each group). b Representative immunocytochemistry images for GFP-tagged BMPRII (in green color) in PAECs treated with either vehicle, BMP-4 (20 ng/mL), or ActA (20 ng/mL) for 30 min. Lysosomes were visualized using LysoTracker (in magenta color). Bars: 20 μm. Similar results were obtained in three independent experiments. c, d Immunoblots and densitometric analysis for BMPRII in PAECs pretreated with either PitStop (PS) (c) or bafilomycin A (BA) (d) for 2 h, followed by treatment with either vehicle or ActA for 30 min in the presence of cycloheximide (n = 3 biologically independent cells in each group). e, f Immunocytochemistry for GFP-tagged BMPRII (in green color) and lysosomes (in magenta color) in PAECs pretreated with either PitStop (e) or bafilomycin A (f), followed by treatment with either vehicle, BMP-4, or ActA. Bars: 20 μm. Similar results were obtained in four independent experiments. g Representative immunoblots and densitometric analysis for serine/threonine-phosphorylated BMPRII in PAECs treated with vehicle, BMP-4, or ActA (n = 3 biologically independent cells in each group). ***P < 0.001; **P < 0.01; *P < 0.05. Exact P values are shown in the Source data file. Data are presented as the mean ± SEM. Two-sided Student’s t-test was used to analyze the differences between the vehicle and ActA-treated groups at each time point (a). One-way ANOVA with Tukey’s post hoc test for multiple comparisons was used to analyze the differences between each study group in all of the western blot quantitation procedures (c, d, and g).