Figure 2
Non-covalent PAR binding of wild-type hTop1, 8bmut and 8hmut. A. The wild-type hTop1, 8bmut and 8hmut were spotted on nitrocellulose membrane and quantified by immunoblot analysis using anti-hTop1 C-terminal antibody or N-terminal-FLAG antibodies. The same graded amounts of proteins were tested for non-covalent PAR binding using radiolabeled PARs. Negative (1 μg DNase I and 1 μg proteinase K, PK) and positive (50 and 25 ng histone H1) (H1) controls have been also spotted on the membrane. The typical ladder of [32P]-PARs is shown on the right and the length of PAR molecules in terms of ADP-ribose residues is indicated. B. Bars represent the ratio of the PAR binding of the mutants and the wild-type protein after normalization with anti-hTop1 (C-terminus) or N-terminal-FLAG antibodies. The values are the means ± SD of three independent experiments.