Physical interactions between RUNX1 and DNA demethylation proteins. (A) Co-IP of endogenous RUNX1 or TET2 followed by western blotting in Jurkat. Input indicates 0.05%, 0.1%, and 1% (from left) of nonimmunoprecipitated cell lysate; IgG, control IP with isotype antibody; IB, immunoblot. (B) HT-based co-IP of HaloTag-fused TET2 (TET2-HT) in TET2-HT and RUNX1 co-overexpressing 293T cells (left) and of HaloTag (HT-ctrl) in HT-ctrl and RUNX1 co-overexpressing 293T cells (right) followed by western blot for RUNX1 protein. (C) HT-based co-IP of HaloTag-fused RUNX1 (RUNX1-HT) in RUNX1-HT and TDG co-overexpressing 293T cells (left), of TET2-HT in TET2-HT and TDG co-overexpressing 293T cells (center), and of HT-ctrl in HT-ctrl and TDG co-overexpressing 293T cells (right) followed by western blotting for TDG protein. (D) HT-based co-IP of RUNX1-HT in RUNX1-HT and GADD45A co-overexpressing 293T cells and of HT-ctrl in HT-ctrl and GADD45A co-overexpressing 293T cells followed by western blotting for GADD45A protein. (E) Schematic representation of RUNX1 deletion mutants. RUNT (red) and TA (light red) denote RUNT DNA-binding and transactivation (TA) domains, respectively. The deleted portions are shown as ranges of amino acid position in the left of each schematic. (F) HT-based co-IP of TET2-HT in TET2-HT and the RUNX1 deletion mutant co-overexpressing 293T cells followed by western blot for V5-tag. The y-axis represents molecular weight. (G) qMSP analysis in RUNX1 deletion mutants overexpressing 293T cells for 3 RUNX1-mediated DNA demethylation target regions (blue) and 2 randomly selected control regions (light blue). Horizontal axes represent methylation percentages with SD. The experiments were performed in 3 biological replicates. WT, wild type.