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. 2024 Aug 7;15:6517. doi: 10.1038/s41467-024-50836-6

Fig. 1. ATM is activated by a defined SSB structure in the HSS system.

Fig. 1

A CTL or SSB plasmid was incubated with HSS at indicated concentrations for 30 min at room temperature. Extracts were examined via immunoblotting. B CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation at room temperature, the extracts were examined via immunoblotting. C CTL or SSB plasmid was added to HSS at different concentrations as indicated. Extracts were examined via immunoblotting analysis. D CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation, the extracts were examined via immunoblotting. E Lambda phosphatase was added to HSS supplemented with CTL or SSB plasmid. After a 15-minute incubation, the extracts were examined via immunoblotting. F ATM inhibitor KU55933 (1 mM), ATR inhibitor VE-822 (1 mM), or DNA-PK inhibitor NU7441 (25 μM) was added to HSS supplemented with CTL or SSB plasmid. After incubation, the extracts were examined via immunoblotting. G CTL or SSB plasmid was added to Mock- or Mre11-depleted HSS. After incubation, the extracts were examined via immunoblotting. H Quantification and statistical analysis of P-ATM/ATM shown in Panel G. Data are presented as mean values ± SD. **p = 0.0011 (two-tailed, paired t-test). n = 3. a.u., arbitrary units. I CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different times of incubation, the extracts were examined via immunoblotting. J Quantification of intensity of protein bands from Fig. 1I& Fig. S1I, and ratios of P-ATM/ATM, and P-Chk1/Chk1 were shown. These samples derive from the same experiment and blots were processed in parallel. Data are presented as mean values ± SD. n = 3. K CTL or SSB plasmid was added to HSS at 40 ng/μL. After different incubation times, the extracts and DNA-bound fractions were examined via immunoblotting. The data presented in Panel A–G, I, and K are representative of three biological replicates.] in Panel C–G indicates mobility shifts of Mre11 and Nbs1.} indicates non-specific bands. Source data are provided as a Source Data file.