Af7646

Cell lysates and immunoprecipitants were used for western blot analysis. Proteins were separated by SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Bio-Rad), and probed with primary antibody. The following antibodies were used: anti-SALL3 (Abnova PAB28233, 1:1000), anti-DNMT3A (Abnova 64B1446, 1:1000), anti-DNMT3B (R&D Systems AF7646, 1:1000), anti-LSD1 (Cell Signaling Technology 2184, 1:1000), and anti-β-actin (Sigma-Aldrich A5441, 1:1000). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, 1:5000), anti-mouse IgG (Invitrogen, 1:5000), or anti-sheep IgG (Abcam, 1:5000). Proteins were visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and ChemiDoc Touch Imaging System (BioRad). Uncropped images of scanned blots shown in Supplementary Figure 12 are provided in Supplementary Information file.

Cells were lysed for 30 min at 4 °C in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) containing complete mini EDTA-free protease inhibitor cocktail (Roche). Cell lysates were clarified by centrifugation for 30 min at 20,000 × g at 4 °C. Cleared lysates were then incubated with antibody for 2 h at 4 °C with rotation. The following antibodies were used: anti-SALL3 (Abnova PAB28233, 1:100), anti-DNMT3B (R&D Systems AF7646, 1:100), rabbit IgG (Cell Signaling Technology 2729, 1:100), and sheep IgG (R&D Systems 5-100-A, 1:100). The lysates were then incubated with protein G magnetic beads (Life Technologies) with rotation for 30 min at 4 °C. The beads were washed three times with lysis buffer. Proteins bound to the beads were eluted using sodium dodecyl sulfate (SDS) sample buffer solution with reducing reagent (Nacalai) at 95 °C for 5 min.