Immunoprecipitation and Western Blot Analysis

Cells were lysed in HEPES immunoprecipitation buffer (10 mM HEPES (pH 8), 2 mM EDTA, 0.1% NP-40) supplemented with 5 mM DTT, 1 mM PMSF, and 1× cOmplete protein inhibitor cocktail (Roche) on ice and subsequently spun down at max speed at 4 °C. The supernatant was removed and protein concentration determined using the Protein Assay Dye Reagent (Bio-Rad). 25 μg protein was removed for input. 500 μg protein was diluted to 1 mg ml⁻¹ in HEPES immunoprecipitation buffer and pre-cleared with 10 μl Protein G magnetic beads (Pierce) for 1 h rotating at 4 °C. Protein lysate was then incubated with 10 μg anti-IgG (Sigma) or anti-POLD1 antibody (Abcam) rotating overnight at 4 °C. The next day, samples were incubated with 30 μl Protein G magnetic beads (Pierce) that had been pre-bound to a bridging antibody (Active Motif) for 1 h rotating at 4 °C. Beads were subsequently washed five times with HEPES immunoprecipiation buffer. Proteins were eluted by incubating beads with 2× sample buffer with BME for 5 min at 95 °C. Samples were analysed by western blot. ChIP was performed as previously described and analysed by western blot and dot blot^{36 (link)}.