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. 2016 Jul 12;44(18):8786–8798. doi: 10.1093/nar/gkw626

Figure 4.

Figure 4.

CDC7 inhibitors affect the phosphorylation levels of cellular TOP2A and its interaction with the kinase. (A) T-REx-EV cells were treated with 10 μM XL413 or DMSO for 3 h, and cell extracts were separated on a 5% SDS-PAGE supplemented with 5 μM Phos-tag acrylamide or on a standard 6% SDS-PAGE. (B) T-REx-DBF4 cells were treated with DMSO or XL413 for the indicated times in the presence of absence of proteasome inhibitor MG132. (C) Extracts prepared from T-REx-DBF4 cells were incubated in phosphatase reaction buffer in the presence or absence of λ-phosphatase. (D) T-REx-DBF4 cells were treated with mimosine for 12 h, then with 10 μM XL413 or DMSO for a further 3 h and immunoprecipitations were performed with anti-TOP2A antibody or control IgG. Immunoprecipiations were perfomed from 1.5 mg of extract and 20 μg of the input material (1.33%) was loaded on the gel. In all cases proteins were analyzed by western blotting; where indicated, low or high intensity scans of the same blot are shown.