Anti dot1l

The primary antibodies used were anti-MLL1 (Bethyl, A300-086A, RRID: AB_242510), anti-MBNL1 (Millipore Sigma, clone 4A8, RRID: AB_10808499), anti-DOT1L (Cell Signalling, D1W4Z, RRID:AB_2799889), anti-SETD1A (Cell Signalling, D3V9S, RRID:AB_2799614), anti-Actin (Cell Signalling Technology, 13E5, RRID: AB_2223172), anti-Lamin B1 (Cell Signaling Technology, D4Q4Z, RRID: AB_2650517) and anti- β-tubulin (Cell Signaling Technology, 9F3, RRID: AB_823664). Whole cell lysates were isolated using RIPA buffer (Sigma) while nuclear extracts were obtained using the NE-PER kit (Thermo Scientific) according to manufacturer’s instructions, and the amount of protein was determined by the BCA Protein Assay Kit (Thermo Scientific). Ten µg of protein was separated by SDS-PAGE on a 4–20% gradient gel (Bio-Rad). After transfer to PVDF membranes, blots were blocked with Odyssey® Blocking Buffer TBS (LI-COR) for one hour and incubated with primary antibodies overnight. After washing, blots were treated with appropriate secondary IRDye 680RD goat anti-mouse (LI-COR) and IRDye 800CW goat anti-rabbit (LI-COR) antibodies at a dilution of 1:10,000 for one hour. Images were obtained using the Odyssey CLx Infrared Imaging System (LI-COR).

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)].