RUNX1 S48, S303, and S424 phospho-specific antisera detect RUNX1 phosphorylation in mammalian cells. (A) Expression of GST-RUNX1(28-86), GST-RUNX1(290-480), and GST-RUNX1(410-480) in bacterial lysates was assessed by Western blotting with GST antibody (left panel). These proteins were incubated with cdk1 in the absence (−) or presence (+) or nonradioactive ATP and again subjected to Western blotting: GST-RUNX1(28-86), with anti–phospho-S48 antiserum (top, right panel); GST-RUNX1(290-480), with anti–phospho-S303 antiserum (center right panel), and GST-RUNX1(410-480), with anti–phospho-S424 antiserum (bottom right panel). (B) 293T cells were transiently transfected with CMV-Myc-RUNX1 (WT) or its S48A, S303A, or S424A variants. Total cell lysates prepared 2 days later were subjected to Western blotting with corresponding phospho-specific antisera (top panels) or c-Myc antibody (bottom panels). (C) Western blotting with phospho-specific or total RUNX1 antisera of extracts obtained from 293T cells transfected with CMV vector (lane 1) or CMV-RUNX1 (lanes 2-3) and treated 24 hours later with 100 μM roscovitine (lane 3), followed by culture for an additional 16 hours.