Cdk1 and cdk6 phosphorylate RUNX1 on S48 and S424 in vitro. (A) Diagram of RUNX1 showing its 3 cdk consensus phosphorylation sites. (B) In vitro kinase assay in which cdk1/cyclin B1 (left panel) or cdk6/cyclin D3 (right panel) were incubated with γ-ATP and GST-RUNX1 fusion proteins containing the RUNX1 amino acid segments 28-290, 290-480, and 290-480 with the S424A mutation, or the 86-217 Runt domain, GST alone, or GST-Rb protein (2 μg), followed by polyacrylamide gel electrophoresis, drying, and autoradiography. (C) In vitro kinase assays (top panels) using the indicated cdk/cyclin complexes and GST-RUNX1 fusion proteins 320-480, 320-480/S424A, 410-480, 410-480/S424A, 27-86, 27-86/T41A, 27-86/S48A, 27-86/T41A/S48A, or GST-Rb. Results are representative of 2 independent assessments for GST-RUNX1(410-480) and its S424A variant and 4 assessments for GST-RUNX1(28-86) and its 3 variants, with the data shown for the right 3 panels from the same experiment run on the same gel (Rb lanes are juxtaposed). Asterisks (*) indicate locations of relevant phosphorylated fragments. Equal volumes of bacterial lysates used for this experiment were also subjected to Western blotting using GST antibody (bottom panels).