Figure 4.

DNA-PKcs phosphorylates hPTTG in vitro. (A) Phosphorylation of hPTTG by DNA-PK. Purified DNA-PKcs–Ku complex was incubated with soluble 6His-fusion proteins in the presence (+) or absence (–) of sonicated DNA (10 µg/ml) and with [γ-³²P]ATP and DNA-PK kinase buffer. Reactions were analyzed by SDS–PAGE and autoradiography. 6His-ΔGMAP was used as control. (B) Phosphorylation of hPTTG by anti-dimer Ku-70/Ku-80 immunoprecipitates. Extracts from HL-60 (10⁷) and HeLa (5 × 10⁶) cells were used to immunoprecipitate the DNA-PKcs–Ku complexes with mAb 162 (anti-dimer Ku-70/Ku-80). After incubation with protein A–Sepharose, the beads were washed and used to perform in vitro kinase assays with soluble 6His-hPTTG and sonicated DNA as described.