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. 2022 Mar 10;29(9):1689–1704. doi: 10.1038/s41418-022-00956-7

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© The Author(s) 2022

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 TUBEs assay for the ubiquitination of PAX9 was conducted in mock-hDPSCs and USP49 KO-hDPSCs using TUBEs-agarose beads. Cell lysates were immunoprecipitated with TUBEs antibodies, followed by immunoblotting with the specific anti-PAX9 antibody. Lane 1, input; Lane 2, mock-hDPSCs; and Lane 3, USP49 KO-hDPSCs. The presented immunoblots are representative of two independent experiments (n = 2). B The ubiquitination and deubiquitination of endogenous PAX9 were analyzed by transfecting hDPSCs with Flag-USP49, Flag-USP49CA, and sgRNA targeting USP49 followed by immunoprecipitation with an anti-PAX9 antibody and immunoblotting with an anti-ubiquitin antibody. The cells were then treated with MG132 for 6 h prior to harvest for all these experiments. The presented immunoblots are representative of two independent experiments (n = 2). C HEK293 cells were transfected with Myc-PAX9, HA-ubiquitin, Flag-USP49, Flag-USP49CA, and sgRNA targeting USP49. The deubiquitination of PAX9 was confirmed by co-immunoprecipitation with the anti-Myc antibody and immunoblotting with the anti-HA antibody. The presented immunoblots are representative of two independent experiments (n = 2). D The TUBEs assay for the ubiquitination of MSX1 proteins was conducted in mock-hDPSCs and USP49 KO-hDPSCs using TUBEs-agarose beads. Cell lysates were immunoprecipitated with the TUBEs antibody followed by immunoblotting with the specific anti-MSX1 antibody. Lane 1, input; Lane 2, mock-hDPSCs; Lane 3, USP49 KO-hDPSCs. The presented immunoblots are representative of two independent experiments (n = 2). E The ubiquitination and deubiquitination of endogenous MSX1 were analyzed by transfecting hDPSCs with Flag-USP49, Flag-USP49CA, and sgRNA targeting USP49, followed by immunoprecipitation with an anti-MSX1 antibody and immunoblotting with an anti-ubiquitin antibody. The cells were then treated with MG132 for 6 h prior to harvest for all experiments. The presented immunoblots are representative of two independent experiments (n = 2). F HEK293 cells were transfected with Myc-MSX1 and HA-ubiquitin, Flag-USP49, Flag-USP49CA, and sgRNA targeting USP49. The deubiquitination of MSX1 was confirmed by co-immunoprecipitation with an anti-Myc antibody and immunoblotting with an anti-HA antibody. The presented immunoblots are representative of two independent experiments (n = 2).