Figure 3

KAP1 Ser473 phosphorylation upon DNA damage. (a) Schematic of human KAP1; known domains are highlighted in color and labeled with bounding amino acid residue numbers. DNA-damage-induced Ser824 phosphorylation site is marked in red. Inset shows an alignment of the region surrounding Ser473 of human [Swissprot: Q13263], mouse [Swissprot: Q62318], and Xenopus laevis KAP1 [Swissprot: Q2TAS5] with the phosphorylated residue highlighted in yellow. (b) KAP1 phospho-Ser473 is detected on western blot after treating cells with various DNA-damaging agents. U2OS cells were not treated (NT) or treated with 1 μM camptothecin (CPT) for 2 h, 5 μM etoposide (ETP) for 2 h, 2 mM hydroxyurea (HU) for 12 h, 10 Gy of ionizing radiation (IR) 1 h before harvesting, 60 μg/ml phleomycin (PHL) for 1 h, or 10 J/m² of ultraviolet light (UV) 1 h before harvesting. (c) Antibodies against KAP1 phospho-Ser473 are specific in immunofluorescence. U2OS cells were transfected with siLuc or siKAP1, irradiated with 20 Gy IR and fixed 2 h afterwards. (d) Specificity of KAP1 phospho-Ser473 antibody by western blotting. U2OS cells stably expressing wild type (wt), S473A, S473D, or S824A versions of GFP-KAP1 were treated with 5 μM etoposide (ETP) for 4 h. Phosphorylation of endogenous KAP1 on Ser824 was used as a DNA-damage readout.