![]() ![]() If not indicated otherwise, proteins were extracted 1 h after end of treatment for western blot analysis. (C-E) Cells were transfected (5 h) with different Dbait molecules as indicated and irradiated (10 Gy) or not. Data represent the mean value and standard deviation of at least three independent experiments. When indicated, inhibitors NU7026 (NU) and wortmannin (Wm) were preincubated with extract 5 min prior to 32H addition (hatched). (B) Stimulation of DNA-PK kinase activity by 20 mM of various Dbait molecules was measured in 1.5 µg HEp-2 nuclear extract. The diamond indicates the supershifted band of Dbait–Ku complexes bound by anti-Ku80. ![]() Antibodies against Ku80 were added prior to migration to confirm by supershift that the bands indicated by asterisks contained Ku80 protein: Lane 1, 32H lane 2, 32H + anti-Ku80 lane 3, 32H + nuclear extract lane 4, 32H + nuclear extract + anti-Ku80. Asterisks indicate one or two dimer shift. (A) Various 32P-labeled Dbait molecules (32H, 24H, 16H, 8H), were incubated with increasing amounts (0-320 ng/ml) of Hep-2 nuclear extract and analyzed on polyacrylamide gels. Hyperactivation of DNA-PK by Double-Strand Break Mimicking Molecules Disorganizes DNA Damage Response Figure 1ĭNA-PK activation and H2AX phosphorylation by Dbait treatment. ![]()
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