Journal: Nature Communications
Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks
doi: 10.1038/s41467-019-09641-9
Figure Lengend Snippet: Peli1 is phosphorylated by ATM that translocate to DNA damage sites. a , b U2OS cells transiently expressing GFP-Peli1 were pre‐treated with ATM (KU55933, ATMi, 10 μM), ATR (VE-831, ATRi, 10 μM), DNA‐PK (KU57788, DNA‐PKi, 10 μM), PARP (PJ34, PARPi, 100 μM), and SUMO (2Dd-08, SUMOi, 50 μM) inhibitors for 1 h. Cells were subjected to laser micro-irradiation. Laser stripes were examined at the indicated time point ( a ). Scale bars, 10 μm. The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( b ). c ATM WT and KO MEF cells were subjected to laser micro-irradiation. Co-immunostaining with γH2AX and Peli1 antibodies at laser-induced DNA lesions (10 min after laser micro-irradiation). Scale bar, 10 μM. d Schematic showing potential Peli1 phosphorylation sites by ATM/ATR kinases. e 293T cells were cotransfected with GFP-Peli1 WT and S121A/T127A mutant. At 48 h post transfection, cells were treated with IR (10 Gy). At 30 min after IR, cells were collected and immunoprecipitated with an anti-GFP antibody. GFP-Peli1 protein complexes were subjected to immunoblotting with anti-pSQ/TQ, anti-GFP, anti-γH2AX, and anti-actin antibodies. f 293T cells were cotransfected with GFP-Peli1 WT and S121A/T127A mutant with or without HA-Ub. At 36 h post transfection, cells were treated with IR (10 Gy) and 30 min later cells were collected and immunoprecipitated with an anti-GFP antibody. GFP-Peli1 protein complexes were subjected to immunoblotting with indicated antibodies. g , h U2OS cells were transfected with plasmids encoding GFP-Peli1 WT or S121A/T127A mutant. At 48 h, cells were subjected to laser micro-irradiation and laser stripes were examined at indicated time point. Scale bar, 10 μM ( g ). The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( h ). i U2OS cells were transfected with plasmids encoding GFP-Peli1 WT or S121A/T127A mutant. At 48 h post transfection, cells were collected and analyzed by immunoblotting with anti-GFP and anti-actin antibodies
Article Snippet: The following reagents were used: ATM inhibitor KU59933 (Tocris), ATR inhibitor VE-831, DNA-PKs inhibitor KU57788 (NU7441), PARP inhibitor PJ34 (Selleckchem, Houston, TX, USA), SUMO inhibitor 2D-08 (Sigma, St. Louis, MO, USA), MG132, cycloheximide, and dimethyl sulfoxide (AG Scientific, San Diego, CA, USA).
Techniques: Expressing, Irradiation, Immunostaining, Phospho-proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot
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