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. 2020 Apr 14;11:1819. doi: 10.1038/s41467-020-15689-9

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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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Fig. 4 — a Protein levels of BRCA2 and pT207-BRCA2 in cells bearing BRCA2 WT or the T207A variant from whole cell lysates of nocodazole-arrested (100 ng/µl for 14 h) or asynchronous cells. 4–15% SDS-PAGE followed by WB using anti-BRCA2. The same blot was stripped and re-probed with anti-pT207-BRCA2 antibody. Lane 3 protein extracts were pre-treated with phosphatase (FastAP) for 1 h before loading onto the gel. Asterisk indicates a non-specific band. (b) GFP-trap pull-down of EGFPMBP-BRCA2 from cells bearing BRCA2 WT, S206C or T207A. 4–15% SDS-PAGE followed by WB using anti-BRCA2 and -PLK1 antibodies. Asynchronous DLD1 cells with endogenous BRCA2 (BRCA2^+/+) were used as control for the pull-down and StainFree images of the gels before transfer as loading control (cropped image is shown). c Quantification of co-immunoprecipitated PLK1 with EGFP-MBP-BRCA2 in (b) relative to the PLK1 protein levels in the input and the amount of pull-down EGFP-MBP-BRCA2 ((PLK1_IP /PLK1_input)/EGFP-MBP-BRCA2_IP) Results are presented as the fold change compared to the BRCA2 WT clone. The data represent the mean ± SD of three independent experiments. One-way ANOVA test with Dunnett’s multiple comparisons test was used to calculate statistical significance of differences (p-values compared to WT). d Top: Synchronization scheme. Bottom: Representative still images of the time-lapse videos. Numbers represent time (min) after nuclear envelope break down (NEBD). Scale bar represents 10 µm. e Quantification of the time the cells spent in mitosis in (d). The red line indicates the median (95% CI). Each dot represents a single cell, n is the total number of cells from two to four independent experiments (45–60 cells per experiment) (BRCA2^+/+ (n = 2), WT C1 (n = 5), BRCA2^−/− (n = 4), S206C A7 (n = 3), T207A B1 (n = 3)). Kruskal–Wallis one-way analysis followed by Dunn’s multiple comparison test was used to calculate statistical significance of differences. f Frequency distribution of the time spent in mitosis in (d), including cells that fail to divide (DEAD). The error bars represent mean ± SD of two to four independent experiments (BRCA2^+/+ (n = 2), WT C1 (n = 5), BRCA2^−/− (n = 4), S206C A7 (n = 3), T207A B1 (n = 3)). Two-way ANOVA test with Tukey’s multiple comparisons test was used to calculate statistical significance of differences. Source data are available as a Source Data file.