Fig. 7: In vitro, S96 phosphorylation has no impact on CST complex formation, CST interaction with RAD51, binding to DNA, or inhibiting MRE11 degradation.

A Co-IP of S96A with CTC1, TEN1, and RAD51 in HEK293T cells co-transfected with Flag-CTC1, HA-TEN1, and MyC-WT-STN1 or Myc-S96A. Cells were treated with 2 mM HU for 3 h. Myc antibody was used for IP. Three independent experiments were performed to ensure reproducibillity. B Purified wild-type CST (WT), C-S96A-T (SA), and C-S96D-T (SD) complexes were resolved in 15% SDS-PAGE and stained with Coomassie blue. Representative result from three independent experiments is shown. C The DNA-binding ability of the CST complex (WT, SA, and SD) was determined by EMSA. The 5′ Cy3- labeled substrates were incubated with the indicated concentrations of CST. Samples were analyzed with 0.8 % agarose gel. Representative result from three independent experiments is shown. D Effects of S96A and S96D on interaction with RAD51 in vitro. Flag-CTC1-STN1-TEN1-His₆ (CST-WT, SA, and SD) was incubated with RAD51, followed by incubation with His-Tag Dynabeads to capture the CST and associated proteins using a magnetic bead separator. The supernatant (S) and eluate (E) were analyzed by 15% SDS-PAGE with Coomassie blue staining. RAD51 alone is shown as a control. Three independent experiments were performed and the result from one experiment is shown. E Effects of S96A and S96D on protecting DNA from MRE11 degradation in vitro. The scheme shows the nuclease activity of MRE11 in degrading 5′ Cy3-labeled substrates (25 nt + 60 nt ssDNA with phosphorothioate bonds on both ends). 5′ Cy3-labeled substrates were pre-incubated with indicated concentrations of CST (CST-WT, SA or SD), then the reactions were completed by adding MRE11. Samples were resolved in 27% denatured polyacrylamide gel. Images show the representative results of 3 independent experiments. The graph represents mean ± S.D (n = 3). Source data are provided in the Source Data file.