Transcriptional and Protein Analysis

Transcriptional assays at individual loci using RT- and RT-qPCR were carried out essentially as in [34 (link)]: primer sequences are listed in Additional file 1: Table S1. Protein was extracted from cells growing in log phase using protein extraction buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton-X, 10% glycerol, 5 mM EDTA; all Sigma-Aldrich) and 0.5 µl protease inhibitor mix (Sigma-Aldrich). For Western blotting, 30 μg protein was denatured in the presence of 5 μl 4× LDS sample buffer (Invitrogen) and 2 μl 10× reducing agent (Invitrogen) in a total volume of 20 μl nuclease-free water (Qiagen) via incubation at 70 °C. Proteins were separated by SDS-PAGE and then electroblotted onto a nitrocellulose membrane (Invitrogen) and blocked in 5% non-fat milk for 1 h at room temperature (RT). Membranes were incubated with anti-DNMT1 (a kind gift from Guoliang Xu) and anti-β-actin (Abcam ab8226) overnight at 4 °C, followed by HRP-conjugated secondary antibody incubation at RT using ECL (Invitrogen).