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. 2015 Jan 20;14(7):1001–1009. doi: 10.1080/15384101.2015.1007001

Figure 6.

Figure 6.

Activation of DNA damage checkpoint pathways prevents the binding of RecQL4, Mcm10, and Ctf4 to replication origins. (A) HeLa cells were irradiated with UV (10 or 20 J/m2), treated with 2 μg/ml 4NQO (4N) or 200 μg/ml zeocin (Ze) for 3 hr. The level of indicated protein in the soluble (SF) or chromatin (CF) fraction was examined by Western blotting. Lane M represented the mock-treated control. (B) The 293T cells stably expressing FLAG-RecQL4 proteins were irradiated with 20 J/m2 UV, and incubated either in the absence or presence of 5 mM caffeine for 3 hr. After preparation of cell extracts, immunoprecipitations were carried out using anti-FLAG M2 agarose beads. FLAG peptide (0.2 mg/ml) was added to whole cell extracts for control immunoprecipitations (Con). (C) HeLa cells irradiated with 20 J/m2 UV were incubated either in the absence or presence of 5 mM caffeine for 3 hr, and the binding of RecQL4, Mcm10, and Ctf4 to replication origins in the Mcm4 upstream promoter region (M4 Ori) or the β-globin locus (β-globin Ori) was examined by ChIP analysis. The primer sets for the 4 kb distal region of M4 Ori and the 9 kb distal region of β-globin Ori were used as internal controls in PCR analyses.