Figure 4. DNA-PKcs phosphorylated on Ser³²⁰⁵ localizes to the midbody in mitosis.

(A) U2OS cells were stained with FITC-conjugated α-tubulin (green) (1:1000 dilution), and a phosphospecific antibody to DNA-PKcs phospho-Ser³²⁰⁵ (red) (1:200 dilution). DNA was stained with DAPI (blue). (B) U2OS cells were treated with 100 nM of the PLK1 inhibitor (BI2536), 100 nM of Aurora-A-Inhibitor-I, 8 μM of the DNA-PK inhibitor (NU7441), or 5 μM of the ATM inhibitor (KU55933) for one hour prior to fixation and stained as above. Also shown as a control experiment (panel 2) in which U2OS cells were stained with FITC-conjugated α-tubulin (green) and the phosphospecific antibody to DNA-PKcs phosphoserine-3205 (red) in the presence of the 10 μg/ml phospho-blocking peptide. DNA stained with DAPI is shown in blue. Additional controls for the specificity of the phospho-Ser³²⁰⁵ phosphospecific antibody are shown in Supplementary Figure S3(C).