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. 2019 Mar 25;10:1368. doi: 10.1038/s41467-019-08911-w

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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. 6 — Trincr1 functions through suppressing TRIM71. a RT-qPCR analysis of Trim71 knocking down in Trincr1−/− ESCs. n = 3 biological replicates. b Western blotting analysis of pERK in control shRNA vector treated wild-type ESCs and control or Trim71 shRNA vector treated Trincr1−/− ESCs cultured in 2i + LIF. Shown are representative images. c Quantification of pERK/ERK. n = 4 independent experiments. d Colony formation assay for control or Trim71 shRNA ESCs in wild type or Trincr1−/− in PD + LIF. n = 3 biological replicates. e Population doubling time for Trincr1−/− and Trim71 knockdown ESCs in 2i + LIF medium. n = 3 biological replicates. The first two columns were the same data appeared in Supplementary figure 1e. f Western blotting analysis and qPCR of Shcbp1 in wild type and Trincr1−/− ESCs. Bottom left: quantification of western blot data, n = 4 independent experiments. Bottom right: qPCR of Shcbp1. Data were normalized to wild-type ESCs. n = 3 biological replicates. g RT-qPCR analysis of Trim71 expression in wild type, Trincr1−/−, and Trincr1_S overexpressing ESCs. Data were normalized to wild-type ESCs. n = 3 biological replicates. h Western blotting analysis of FLAG knockin TRIM71 in wild type, Trincr1−/−, and Trincr1_S overexpressing ESCs in 2i + LIF. n = 3 independent experiments. i Western blotting analysis of immunoprecipitation with FLAG antibody (FLAG IP) in HEK293 cells expressing HA-SHCBP1 and FLAG-TRIM71 with or without Trincr1_S overexpression. Shown are representative images of IP samples (left) and quantification of IP efficiency (right). IP efficiency was calculated as HA-SHCBP1 /FLAG-TRIM71 and then normalized to samples without Trincr1_S overexpression. n = 3 independent experiments. For the RT-qPCR of a, f, and g, the β-actin gene was used as a control. Shown are mean ± SD. For a, c, and d, data were normalized to wild-type ESCs treated with control shRNA vectors, P values were determined by unpaired one-way ANOVA with two-sided Dunnett’s test. For e, P-values were determined by unpaired two-way ANOVA with Tukey’s test. For f and i, P values were determined by paired two-sided Student’s t-test