Fig. 1: CST and RPA localize in close proximity on ssDNA in cells in response to replication stress.
From: Crosstalk between CST and RPA regulates RAD51 activity during replication stress
a Crosstalk among CST, RPA, and RAD51 on ssDNA subjected to replication stress. b CST lies in close proximity to RPA upon fork stalling. (i) PLA is a technique that detects the physical proximity of two different proteins. In principle, if the two proteins are <40 nm apart, fluorescence signal can be detected. In brief, the two target proteins are bound by specific primary antibodies. If the target proteins are sufficiently proximal, PLA secondary antibodies hosting oligonucleotides can be ligated by means of two PLUS/MINUS PLA oligos to circularize. The DNA polymerase phi29 then processes rolling-circle amplification, and the resulting copies can be detected by hybridizing the fluorescence-labeled oligonucleotide51. (ii), (iii) PLA assays were performed to establish the close proximity of CTC1/STN1 with RPA in HeLa cells treated with hydroxyurea (HU) for 3 h. Representative PLA images of CTC1/RPA32 (ii) and STN1/RPA32 (iii) are shown. Scatter plots from one experiment are shown here. Red lines represent mean values ± SEM. N: the number of cells analyzed in each sample. P values were calculated by one-way ANOVA. NS not significant, ***P < 0.001; ****P < 0.0001. c CST colocalizes with RPA on the same ssDNA in response to fork stalling. HeLa cells expressing Flag-CTC/Myc-STN1/HA-TEN1 were labeled with BrdU and treated with or without HU (3 h), followed by co-immunostaining with anti-Flag (red), anti-RPA32 pS33 (cyan), and anti-BrdU (green) antibodies. N = 3 biologically independent experiments.
