Ab113474 nuclear extraction kit

30.000 cells were plated in 6 well plates 24 h before treatment. Drug treatment was performed for 24 h and nuclear extracts of treated cells were prepared according to the instructions of the manufacturer (ab113474 – Nuclear Extraction Kit, abcam, Cambridge, UK). The, KDM4 activity was determined in nuclear extracts by a fluorescence-based method according to the manufacturer’s instructions (ab113462 –KDM4/JMJD2 Activity Quantification Kit, abcam, Cambridge, UK).

Total protein extractions were obtained using lysis solution and cytoplasmic and nuclear protein fraction extractions were obtained using ab113474 Nuclear Extraction Kit (Abcam). Protein lysates were separated on a 12% SDS-PAGE gel prepared with 40% Acrylamide–Bisacrylamide 29:1 (Invitrogen, Carlsbad, CA, USA) and 4X separating buffer (Alfa Aesar, Ward Hill, MA, USA) for the separating gel or 4X stacking buffer (Alfa Aesar, Karlsruhe, Germany) for the stacking gel. After run the proteins were blotted onto a nitrocellulose membrane of the iBlot^® gel transfer stacks (Kiryat Shmona, Israel) using the iBlot™ dry transfer system (Life Technologies, Herzliya, Israel). The primary and secondary antibodies used are listed in Supplementary Table S3. The assessment of the bands’ density was made in the ImageLab software (BioRad) using the measures of Tubulin (for total and cytoplasmic protein fractions) and HDAC1/Lamin B1 (for nuclear protein fraction) as loading controls. A representative Western blot of the purity of nuclear and cytoplasmic protein fractions extraction is shown in Supplementary Figure S1.