Rabbit anti phospho histone h3 ph3

After 48 h of transfection with pcDNA‐NR2F1‐FLAG (WT or pathogenic variants), HEK293T cells were fixed with 70% cold ethanol for 15 min and then washed with 3 mL of PBS containing 1% FBS. The fixed cells were blocked with a pre‐blocking solution (PBS, 0.3% tween, 5% serum) and then incubated for 30 min at 4°C with gentle shaking. The cells were co‐stained with primary antibodies, mouse anti‐NR2F1 (1:1000) (R&D), rabbit anti‐phospho‐Histone H3 (pH 3) (1:1000) (Millipore), or rabbit anti‐cleaved Caspase 3 (1:1000) (Cell signaling) and incubated for 2 h at 4°C with gentle shaking. After washing, cells were stained with Alexa Flour 488 anti‐mouse and Alexa Flour 647 anti‐rabbit antibodies for 1 h at 4°C with gentle shaking. Then the cells were washed with PBS. To study the cell cycle, cells were stained with propidium iodide (PBS, 0.1% NP40, 0.2% RNase, 3.95% propidium iodide) for 15 min at RT followed by two washes with PBS. After filtering through a 70‐micron filter, the cells were analyzed by FACS using a BD LSRFortessa and FACSDiva software (Becton Dickinson). Cells were analyzed on the basis of 10.000 total events (debris excluded) per experiment; for cell cycle and apoptosis analyses, cell percentages were calculated over the number of NR2F1+ cells and normalized on the control sample (cells transfected with full‐length WT NR2F1). All antibodies used are listed in Table S6.