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. 2018 Jan 19;46(5):2479–2494. doi: 10.1093/nar/gky020

Figure 2.

Figure 2.

An ATR–Chk1 DNA damage response pathway is induced by the defined SSB structure in the HSS system. (A) CTL or SSB plasmid was added to HSS at different concentrations as indicated, and incubated for 30 min. Extracts were examined via immunoblotting analysis for Chk1 phosphorylation (i.e., Chk1 P-Ser344) and total Chk1. (B) CTL or SSB plasmid was added to HSS at a final concentration of 75 ng/μl. After different time of incubation at room temperature, the extracts were examined via immunoblotting analysis. (C–E) ATR inhibitor VE-822, ATM inhibitor KU55933, DNA-PK inhibitor NU7441, recombinant geminin, or DNA polymerase inhibitor Aphidicolin was added to HSS supplemented with CTL or SSB plasmid at a final concentration of 75 ng/μl for 30 min. Extracts were examined via immunoblotting analysis as indicated. (F) Geminin or roscovitine was added to HSS supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, extracts were examined via immunoblotting analysis as indicated. (G) CTL, SSB or DSB plasmid was added to HSS at different concentrations as indicated. Samples were examined via immunoblotting analysis.