FIG. 8.

Effect of BrD affinity depletion or affinity competition on hGCN5 phosphorylation in the P11 FT fraction. (A) Outline of experiments testing effect of GST-BrD on hGCN5 phosphorylation. P11 column FT was tested for Ku70-dependent effects on hGCN5 phosphorylation, following either affinity depletion of Ku70 or affinity competition for Ku70, using GST-BrD. The experimental results are shown in panels B (depletion) and C (competition). (B) Effect of affinity depletion of Ku70 on phosphorylation of hGCN5. The P11 FT was either not treated (−) or incubated with GST-BrD beads or GST beads. Proteins bound to the beads were depleted by centrifugation. Upper panel, [γ-³²P]ATP and sonicated salmon sperm DNA were added to the supernatants to activate DNA-PKcs. Following the kinase reaction, samples were immunoprecipitated using α-hGCN5 and subjected to SDS-PAGE and autoradiography. Middle and lower panels, following bead depletion, either the supernatants were assayed for hGCN5 (middle) or the beads were assayed for Ku70 (lower), using Western analysis. (C) Effect of affinity competition for Ku70 on phosphorylation of hGCN5. GST-BrD and GST were eluted from the glutathione-Sepharose beads, using reduced glutathione. The P11 FT was incubated with the bead-eluted GST-BrD or GST, and a kinase assay was performed as for panel B.