Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Apr 24;11:1972. doi: 10.1038/s41467-020-15879-5

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Fig. 5 — a Gel filtration profiles show the interaction between REV7^WT/R124A-SHLD3(1–82) (short as WT/R124A-1–82 in the Figure) and MBP-SHLD2(1–60) in a Superdex200 Increase 10/300 size exclusion chromatography (SEC) column (the left panel). MBP-SHLD2(1–60) is excessive. The peaks eluted at 12.5–13 ml are composed of the stable complex of MBP-SHLD2(1–60)-REV7-SHLD3(1–82) while the peaks eluted at 14.5–15 ml are un-complexed MBP-SHLD2(1–60) or REV7-SHLD3(1–82). Fractions (0.5 ml each) corresponding to REV7^WT-SHLD3(1–82) and MBP-SHLD2(1–60) co-elution (black), REV7^R124A-SHLD3(1–82) and MBP-SHLD2(1–60) co-elution (red) were analyzed by SDS-PAGE and stained by Coomassie brilliant blue (the right panel). (n = 2). b MBP pulldown showing the interaction between MBP-SHLD2(1–60) and REV7^WT/R124A-SHLD3(1–82). Samples were analyzed by SDS-PAGE and stained by Coomassie brilliant blue. (n = 4). c Gel filtration profiles show the interaction between REV7 mutants and MBP-SHLD2(1–60). Various mutants of REV7-SHLD3(1–82) complex (200 μg) were first incubated with or without REV7^K129A (short as K129A in the Figure) (200 μg), after 10 min, excessive MBP-SHLD2(1–60) was added. Fractions (0.5 ml each) corresponding to REV7^E35A-SHLD3(1–82)-REV7^K129A (short as E35A-1–82 + K129A in the Figure) and MBP-SHLD2(1–60) co-elution (black), REV7^K44A-SHLD3(1–82)-REV7^K129A (short as K44A-1–82 + K129A in the Figure) and MBP-SHLD2(1–60) co-elution (red), REV7^K129A-SHLD3(1–82)-REV7^K129A (short as K129A-1–82 + K129A in the Figure) and MBP-SHLD2(1–60) co-elution (blue), REV7^E35A-SHLD3(1–82) (short as E35A-1–82 in the Figure) and MBP-SHLD2(1–60) co-elution (pink) were analyzed by SDS-PAGE and stained by Coomassie brilliant blue (the right panel). (n = 2). d MBP pulldown assay shows the interaction between MBP-SHLD2(1–60) and REV7-SHLD3(1–82) mutants or reconstituted conformational dimer. Samples were analyzed by SDS-PAGE and stained by Coomassie brilliant blue. (n = 2). e NHEJ efficiency was determined in Hela cells overexpressing exogenous FLAG-tagged wild-type REV7, REV7-K44A, REV7-K190A or control vector. Data were analyzed with the unpaired two-tailed Student’s test. Error bars indicate SEM (n = 3). Ns, no significant difference, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p-values are 0.0011 (Control versus REV7-K190A), 0.00024 (REV7-WT versus REV7-K190A), 0.000093 (REV7-K44A versus REV7-K190A), 0.22 (Control versus REV7-WT), 0.36 (Control versus REV7-K44A) and 0.54 (REV7-WT versus REV7-K44A). n, biologically independent experiments. Source data are provided as a Source Data file.