Anti h3k79me2

Protein extracts were always prepared from L4 animals (except when comparing 1-d-old adult and 4-d-old adults). Samples were boiled in SDS–PAGE buffer for 2 × 5 min and sonicated for 10 min using a Diagenode Bioruptor (UCD-300). The following antibodies were used: anti-H3K4me3 (C42D8; 1:750; Cell Signaling Technology); anti-H3K4me1 (ab8895; 1:1,000; Abcam); anti-H3K4me2 (07-030; 1:1,000; Millipore) anti-H3 (ab1791; 1:10,000; Abcam); anti-H3K9me3 (ab8898, 1:1,000; Abcam); anti-H3K27me3 (MABI0323, 1:1,000; Thermo Fisher Scientific); anti-H3K36me3 (D5A7; 1:1,000; Cell Signaling Technology); anti-H3K79me2 (D15 × 10⁸; 1:1,000; Cell Signaling Technology) and peroxidase-labelled anti-rabbit and anti-mouse secondary antibodies (1:10,000; Vector Laboratories). Western blots were quantified using ImageJ (National Institutes of Health).

Total cell lysates were prepared in 2× sample loading buffer [250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad, Hercules, CA, USA]. The protein concentrations of samples were quantified using the bicinchoninic acid (BCA) method [34 (link)] and a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (5–20 μg) were separated by 6–13% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) and then probed with anti-DOT1L, anti-H3K79me2, anti-β-Actin, anti-histone H3, anti-H3K4me2, anti-H3K27me2, anti-H3K36me2, anti-H3K79me1, anti-H3K79me3, and anti-Vimentin antibodies purchased from Cell Signaling Technology (Beverly, MA, USA) or with anti-E-cadherin and anti-N-cadherin antibodies purchased from (BD Biosciences, San Jose, CA, USA). The blots were detected using a WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Korea) [35 (link)].