RUNX1 S48, S303, and S424 phosphorylation and cell-cycle variation in a hematopoietic cell line. (A) Ba/F3 cells expressing RUNX1 from the MT promoter were cultured in zinc chloride for 16 hours and then subjected to density fractionation by elutriation. An aliquot of each fraction was stained with Hoechst 33258 dye followed by FACS analysis to allow estimation of the percentage of cells in G1, S, or G2/M, as shown. (B) Total cell extracts corresponding to 2 × 106 cells were subjected to Western blotting. The blot was stained sequentially with anti–phospho-S48, anti–phospho-S303, anti–phospho-S424 antisera, and anti-RUNX1 antibody and was also stained with Fast Green to confirm equivalent loading. Relative expression in fraction 30 compared with the mean of fractions 20 and 22 is shown to the right of each panel. (C) Cycloheximide (CHX) was added to 293T cells 24 hours after transfection with CMV, CMV-RUNX1, CMV-RUNX1(tripleA), or CMV-RUNX1(tripleD), and total cell proteins prepared 0, 4, 8, or 16 hours after CHX addition were subjected to Western blotting using anti-RUNX1 antiserum. The blot was also stained with Fast Green.
