Subcellular localization of the WT and mutated RUNX1 proteins. (A) Schematic representation of RUNX1 showing location of functional domains and specific RUNX1 mutations used in our study. Horizontal bars indicate RUNX1 (453 aa), including the RHD domain (50-177 aa), the ES domain (242-262 aa), the TAD domain (291-371 aa), and the TRD domain (371-411 aa). The numbers in the left column indicate the unique patient numbers (UPN2, UPN4-UPN8, UPN10, and UPN11) described in Table 1. Mutant no. in the right column indicates the number of mutants. (B) Subcellular localization of the indicated RUNX1 proteins was visualized by confocal microscopy analysis in the 293T cell line. Left panels show the merged images; middle panels, localization patterns of WT RUNX1 or mutants; and the right panels show nuclei as visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. (C) Expression patterns of RUNX1 mutant proteins in the 293T cell line. A total of 2 × 106 cells for each mutant were used for stepwise separation of cytoplasmic and nuclear extraction, and extracted proteins from an equivalent of 4 × 105 cells per fraction were used for WB. An Ab against RUNX1 was used to detect the exogenously expressed RUNX1 proteins. The 2 different gels are indicated by the gray dividing lines; n indicates nuclear protein extraction; and c, cytoplasmic protein extraction.
