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. 2020 Dec 2;40(2):e104542. doi: 10.15252/embj.2020104542

Figure EV4. DNA damage‐induced Ser139 phosphorylation does not affect ADP‐ribosylation on H2AX.

Figure EV4

  1. 293T cells were pre‐treated with pan PI3‐like kinase inhibitor (Torin2: 0.5 µM for 1 h) followed by H2O2 stimulation (2 mM in PBS, 5 min). 0.1% DMSO in medium for 1 h as mock. γH2AX was examined by Western blotting using anti‐γH2AX antibody. Actin was blotted as a control.
  2. 293T cells were pre‐treated with pan PI3‐like kinase inhibitor (Torin2: 0.5 µM for 1 h) followed by H2O2 stimulation (2 mM in PBS, 5 min). 0.1% DMSO in medium for 1 h as mock. ADP‐ribosylated proteins were immunoprecipitated with anti‐ADPR antibody; ADP‐ribosylated H2AX was examined by Western blotting using anti‐H2AX antibody.
  3. The Ser139 phosphorylation does not affect the H2AX ADP‐ribosylation. The cells expressing the wild‐type H2AX, the S139A mutant, and the E141A mutant were treated by H2O2 stimulation (2 mM in PBS, 5 min) and then examined by IP and Western blotting with indicated antibodies.