Thermo scientific ne per nuclear and cytoplasmic extraction kit

Nuclear proteins from kidney marrow cells were extracted using the Thermo Scientific™ NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the instructions of the manufacturer. After extraction, these proteins were electrophoresed on Mini-PROTEAN^® TGX Stain-Free™ Precast 10% gels (Bio-Rad, Hercules, CA, USA), transferred onto a 0.2 µm PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA), and processed using standard methods. As primary antibodies, rabbit anti-human RUNX1 antibody (1:400 in 5% BSA, ab 23980, Abcam, Cambridge, UK) and rabbit anti-β-Actin (1:60,000 in 5% BSA, sc-1615-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. As a secondary antibody, the goat anti-rabbit IgG antibody was conjugated to Horseradish Peroxidase (1:7000 in 5% BSA, #7074S, Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence was initiated with Cell Amersham ECL Reagent (GE Healthcare, Chicago, IL, USA) and detected using ChemiDocTM XRS+ with Image LabTM Software (version 6.0.1, Bio-Rad, Hercules, CA, USA). Protein sizing relied on the PageRuler Plus prestained ladder (Thermo Fisher Scientific, Waltham, MA, USA), and image processing was performed with Fiji (ImageJ, v. 1.59i).