Fig. 5: RIF1-L-associated PP1 suppresses S2265 phosphorylation to limit RIF1-L-BRCA1 interaction.

A, B RIF1-BRCA1 co-IP analysis. Flp-In T-REx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. FLAG IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. C RIF1-BRCA1 co-IP analysis. Reciprocal co-IP to (A, B). Flp-In T-REx 293 cells expressing indicated GFP-RIF1 constructs were transfected with FLAG-BRCA1 plasmid. GFP IP was performed and immunoblotted for GFP-RIF1 and FLAG-BRCA1. D RIF1-BRCA1-BRCT co-IP analysis. Flp-In T-REx 293 cells expressing indicated GFP-RIF1 constructs were transfected with mCherry-BRCA1-BRCT plasmid. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging. E Schematic representation of RIF1 constructs used in (D) and their co-IP analysis outcomes with BRCA1-BRCT. F Western blot analysis of RIF1-L-Phospho-S2265 signal in HeLa RIF1 KO cells supplemented with Dox-inducible RIF1-L or RIF1-L-pp1bs. (HeLa cell characterisation presented in Supplementary Fig. 3E). G Representative images of RIF1-BRCA1 PLA foci in HeLa RIF1 KO cells (left) and in RIF1 KO cells supplemented with RIF1-L (middle) or RIF1-L-pp1bs (right). HU: 4 mM 24 h. Scalebar: 10 µm. H Quantification of RIF1-BRCA1 PLA foci number per nucleus in cells from the experiment as (G). p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. I RIF1-BRCA1-BRCT co-IP analysis. Flp-In T-REx 293 cells expressing GFP-RIF1-L-pp1bs or GFP-RIF1-L were transfected with mCherry-BRCA1-BRCT plasmid. 16 h after transfection, cells were further treated with no, or 4 h, or 24 h of 4 mM HU. mCherry IP was performed and immunoblotted for GFP-RIF1 (top panels). Lower panels show protein visualised by stain-free gel imaging.