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. 2019 Mar 6;7:8. doi: 10.1038/s41413-019-0046-y

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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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Fig. 5 — COL2A1 repressed BMP-SMAD1 signaling activation through facilitating the ITGB1−SMAD1 interaction and weakening the BMPR1A/B−SMAD1 interaction. a, b Immunoprecipitation was performed on 293T extracts with anti-SMAD1 antibody or anti-ITGB1 antibody, followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. c 293T cells were transfected with SMAD1-Flag, ITGB1-HA or vector-Flag plasmids, and immunoprecipitation was carried out with anti-Flag antibody followed by immunoblotting with anti-HA antibody and anti-Flag antibody. d 293T cells were transfected with SMAD1-Flag, ITGB1-HA, or vector-HA plasmids, and immunoprecipitation was carried out with anti-HA antibody, followed by immunoblotting with anti-HA antibody and anti-Flag antibody. e SMAD1 protein was purified from 293T cell extracts expressing SMAD1-Flag and identified by Coomassie blue staining (left panel) and mass spectrometry (right panel). f Purified SMAD1-Flag protein was incubated with the product expressing ITGB1-HA, which had been linked to HA affinity agarose beads for 4 h at 4 °C, followed by bead washing and immunoblotting. g, h Immunoprecipitation was performed on 293T cells treated with 100 μg·mL⁻¹ COL2A1 or vehicle (0.05 mol·L⁻¹ acetic acid) for 1 h with anti-SMAD1 antibody (g) or anti-ITGB1 antibody (h), followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. i, j Immunoprecipitation was performed on 293T cells treated with COL2A1 or vehicle with anti-SMAD1 antibody (i) or anti-BMPR1A antibody (j), followed by immunoblotting with anti-SMAD1 antibody and anti-BMPR1A antibody. k, l Immunoprecipitation was performed with anti-SMAD1 antibody (k) or anti-BMPR1B antibody (l) on 293T cells treated with COL2A1 or vehicle followed by immunoblotting with anti-SMAD1 antibody and anti-BMPR1B antibody. m, n 293T cells were pretreated with 10 μg·mL⁻¹ ITGB1 blocking antibody or anti-human IgG antibody for 1 h and then treated with COL2A1 or vehicle for 1 h. Immunoprecipitation with anti-SMAD1 antibody (m) or anti-ITGB1 antibody (n) was conducted, followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. Blk ab, blocking antibody