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. 2020 Aug 6;10:13275. doi: 10.1038/s41598-020-70289-3

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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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PRMT1-dependent methylation of BRCA1 controls the stability and localization of Bcl-2. (A) Coimmunoprecipitation of BRCA1 and Bcl-2 in MCF7 cells (IR, 10 Gy). Total extracts (1 mg) were immunoprecipitated using anti-BRCA1 (clone 6B4). Samples from different experimental conditions were processed in parallel and subjected to the same exposition time and image conditions (Fig. S9). The histogram represents coimmunoprecipitated Bcl-2 with respect to input Bcl-2 in siControl and siPRMT1 cells and relative to nonirradiated cells. The IR-dependent decrease in Bcl-2 was only statistically significant in siControl cells (P < 0.005 at all assayed times). Effective silencing was determined in the inputs (right panels). (B) Confocal microscopy showing Bcl-2 localization in MCF7 cells. Mitochondria were stained with MitoTracker-Red CMXRos. Bar represents 5 μm. BRCA1 silencing was evaluated (right panel; bars, 15 μm). (C) The effects of PRMT1 or BRCA1 silencing on Bcl-2 protein and mRNA levels in MCF7 cells in the absence or the presence of MG132 (10 µM; 10 h). Decrease of Bcl-2 mRNA was only significant in siBRCA1 cells when compared with siControl cells. (D) Effect of IR (10 Gy; 5 h) on Bcl-2 protein levels in the indicated MCF7 cells (*P < 0.05). (E) The ubiquitinated state of Bcl-2 in MCF7. Samples from different experimental conditions were processed in parallel and subjected to the same exposition time and image conditions (Fig. S9). The histograms represent ubiquitinated Bcl-2 with respect to immunoprecipitated Bcl-2 and relative to ubiquitinated Bcl-2 in nonirradiated siControl cells. The IR-dependent decrease in ubiquitinated Bcl-2 was only statistically significant in siControl cells (P < 0.005 at all assayed times). Silencing of PRMT1 or BRCA1 was tested in the inputs and results were similar as observed in A. (F) Western blot showing poly-ubiquitylated forms of Bcl-2 in MCF7 cells after indicated siRNA transfections. Equal amounts of Bcl-2 per lane were loaded. Silencing of PRMT1 and/or BRCA1 was tested in the inputs (similar results as observed in Fig. 5A). The groupings blots in this figure were cropped from different gels. Full blots are shown in the Supplementary Information, Fig. S9.