FIG. 4.

Mutation of the cluster of CK2 phosphorylation sites at S518/T519/T523 greatly reduces or ablates interaction with PNK. (A) Total cell extracts from 5 × 10⁴ of cells expressing the indicated recombinant XRCC3 protein or harboring empty vector (v) were fractionated by SDS-PAGE, transferred to nitrocellulose, and immunostained with anti-XRCC1 polyclonal antibody or anti-β-actin monoclonal antibody. (B) XRCC1-His protein complexes were recovered from whole-cell extract from 5 × 10⁶ of the indicated cells by affinity chromatography as described in Materials and Methods. Aliquots of the column input (8% of column input), flowthrough, and 250 mM imidazole eluate (13% of total eluate) were fractionated by SDS-PAGE and immunoblotted for the presence of anti-XRCC1 (αXRCC1) or anti-PNK (αPNK) polyclonal antibody. (C) Clonogenic sensitivity of the indicated cell lines to H₂O₂. Cells were treated for 15 min at room temperature with the indicated concentrations of H₂O₂ in PBS, and surviving colonies were stained and counted after 10 to 14 days. Data are the means of at least three independent experiments (± standard errors of the means).