Phospho-proteome Analysis of Irradiated Cells

For analysis of the phospho-proteome, cells were plated and irradiated with 10 Gy SD, or with 10 fractions of 1 Gy dose per fraction with two fractions per day (Fig. 1A). At 30 min (ST) and at 2 months (LT) after irradiation, the cells were lysed from plates in T-Per (ThermoFisher Scientific) mixed 1:1 with 2X SDS Tris–Glycine buffer (Invitrogen, Carlsbad, CA) + 2-mercaptoethanol (final concentration = 2.5%). Reverse phase protein microarrays were performed as previously published^{22 (link)}. In brief, samples were diluted and printed in duplicates onto nitrocellulose slides. HeLa cell lysates (with or without pervanadate) were used as positive and negative controls (Supplementary Figure S2). Microarrays were stained with specific and validated antibodies and analyzed with a biotin-linked signal amplification system (DAKO). The total protein amount of the sample was determined with the SYPRO Ruby stain (ThermoFisher Scientific).