The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β  MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.