Fig. 6: S1578 phosphorylation mediated by ATM stabilised CBP.

Control (shSCR) and ATM knockdown (shATM) MOLM-13 (A) and MV4-11 (B) cells were treated with 25 μM MG132 for 4 h. The cell lysates were subjected to IP with CBP antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. C 293T cells were transfected with HA-CBP or Flag-ATM, and HA-CBP or Flag-ATM was purified. Purified HA-CBP was subjected to in vitro phosphorylation with or without Flag-ATM, and the reactions were then detected by western blotting with the indicated antibodies. D Wild-type (WT) CBP and different HA-tagged CBP (HA-CBP) mutants (S718A, S1578A, T1697A, and S1755A) were transfected into 293T cells. After 72 h, the cell lysates were subjected to IP with HA antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. E The WT, S1578A and AAAA (S718A/S1578A/T1697A/S1755A) HA-CBP were transfected into 293T cells. After 72 h, the cell lysates were subjected to IP with HA antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. F WT and S1578A HA-CBP with or without Flag-ATM were transfected into 293T cells. 72 h later, the cell lysates were subjected to IP with HA antibodies. The Input and IP samples were then detected by western blotting with the indicated antibodies. G 293T cells were transfected with WT or S1578A HA-CBP, Flag-ATM. HA-CBP or Flag-ATM was purified. Purified HA-CBP was subjected to in vitro phosphorylation with or without Flag-ATM, and the reactions were then detected by western blotting with the indicated antibodies. H Purified HA-CBP was subjected to in vitro dephosphorylation with Lambda PP at 30 °C for 3 h. Dephosphorylated HA-CBP was then subjected to in vitro phosphorylation with or without Flag-ATM, and the reactions were detected by western blotting with the indicated antibodies. I WT, S1578A, and S1578E HA-CBP were transfected into 293T cells. At 36 h posttransfection, the cells were treated with 25 μM MG132 for 20 h. The cell lysates were subjected to IP with HA antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. MOLM-13 (J, K) and MV4-11 (L, M) cells were transduced with lentivirus expressing wild-type (WT) or mutant HA-CBPΔ (S1578E, S1578A). The cells were incubated with 40 μg/ml CHX for the indicated times. The total lysates of the cells were then subjected to western blotting with the indicated antibodies. The band intensity of HA was normalised to that of ACTB in the same sample, and each time point from three independent experiments was normalised to time 0 for statistical analysis. The difference in slopes between every two groups was compared. ns not significant. ***P < 0.001.