SYCE2 potentiates the steady-state ATM activity.

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SYCE2 potentiates the steady-state ATM activity. SYCE2 potentiates the steady-state ATM activity. (A, B) Immunoprecipitation/Western blot analysis showing the levels of autophosphorylation of ATM on Ser 1981 and ATM expression. 500 μg of total cell lysates of mock cells and RPE cells expressing FLAG-SYCE2 (A) or MCF7 cells transfected with a nontargeting siRNA control or SYCE2-targeting siRNA (B), unirradiated or irradiated with 8-Gy x-ray 30 min before harvesting, was precipitated using the anti-ATM antibody or normal rabbit IgG and visualized by Western blotting using the anti-phospho-ATM (Ser 1981) antibody and the anti-ATM antibody. 30 μg of each lysate was also subjected to Western blot analysis using the anti-FLAG-antibody and anti-CDK2 antibody. Please note that the membranes were not reprobed because the band of ATM is not distinguishable from that of phosphorylated ATM. (C) Immunofluorescence visualization of foci of ATM phosphorylated on Ser 1981 (green) in MCF7 cells transfected with nontargeting control siRNA (upper panel) and in MCF7 cells transfected with SYCE2-targeting siRNA (lower panel), stained at 10 min after 0.2-Gy x-ray irradiation. Scale bar, 20 μm. (D, E) Percentages of cells containing three or more large phospho-ATM (Ser 1981) foci. Columns and bars represent the mean of three independent experiments and SD, respectively. A total of 100 cells were examined for each cell line. (F, G) Sensitivities of the mock cells and FLAG-SYCE2–expressing RPE cells treated with either DMSO alone or 10 μM KU-55933 dissolved in DMSO for 24 h before x-ray irradiation (F) and 1 h treatment with cisplatin (G). Noriko Hosoya et al. LSA 2018;1:e201800021 © 2018 Hosoya et al.