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 May 11;219(6):e201908192. doi: 10.1083/jcb.201908192

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

© 2020 Duan et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 8. — SMARCAL1-mediated fork reversal is required for accumulation of hyperubiquitinated RPA in BRCA2-deficient cells. (A) Western blot-based analysis of pRPA32 accumulation after disrupting reversed fork formation by codepletion of SMARCAL1 in BRCA2 deficient cells. Experimental conditions used are as described above. Nuclear extracts were prepared. Blot was probed with anti-pRPA32 (S4/S8). (B and C) IF and graphs of pRPA32 (S33) recruitment in U2OS cells transfected with the indicated siRNAs. Cells were treated with 5 mM HU and harvested 4 h after damage. ***, P < 0.005. Statistical significance was determined by the two-tailed Student’s t test and the error bars indicate SD (n = 3). Scale bars in B indicate 10 µm. (D) Immunoprecipitation analysis of RPA ubiquitination in HEK293T cells transfected with indicated siRNAs. Experimental conditions used are as described above. (E) Quantification of IF-based analysis of 53BP1 foci in U2OS cells transfected with indicated siRNAs. Experimental conditions used are as described above. Graph indicates the percentage of cells with >10 53BP1 foci per cell. ***, P < 0.005. (F) CellTiter-Glo–based cell survival assay was used to determine the sensitivity of U2OS cells to HU in cells depleted of BRCA2, SMARCAL1, and/or BRCA2+SMARCAL1. Error bars indicate SD between triplicates. Experimental conditions used are as described above.