Subcellular Fractionation and Histone Mark Detection

A subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific, Waltham, MA, USA; 78840) was used for fractionation experiments according to the manufacturer’s instructions. A 10 cm dish format was applied, which corresponded to a packed cell volume of 20 µL per well. Localization of SAMD1 was determined using a homemade SAMD1 antibody recognizing the SAM domain [9 (link)]. As loading controls for the respective fractions, a homemade SP1 antibody [18 (link)], anti-tubulin (Merck, Kenilworth, NJ, USA; MAB3408), and anti-H2Aub (Cell Signaling Technology, Danvers, MA, USA; 8240) were applied. For the detection of histone marks, the chromatin-bound fraction was used. H3 (Abcam, Cambridge, UK; ab1791), H3K4me2 (Diagenode, Denville, NJ, USA; C15410035), H3K4me3 (Diagenode, Denville, NJ, USA; C15410003) and H3K14ac (Abcam, Cambridge, UK; ab52946) antibodies were applied. The signal was quantified using the ImageLab software (v5.2.1, Bio-Rad, Hercules, CA, USA) and normalized to the H3 signal.