Physical and functional interaction of Id1 with AKT1 in leukemia cells. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie staining Kasumi-1-MIGR1-HA-Id1 cells using an anti-HA antibodies and normal mouse immunoglobulin (Ig) G as control. (Asterisk identifies a differentially expressed sodium dodecyl sulfate–polyacrylamide gel electrophoresis band.) (B) Immunoprecipitation was performed using anti-Id1 antibody in the Kasumi-1 cells treated with CBD or vehicle control, and IgG was used as control. The anti-AKT1 and anti-Id1 antibodies were used for western blot analysis. (C) Id1 interacts with AKT1 in vitro. Purified GST-Id1 and His-AKT1 were used for GST pull-down assays, and anti-AKT1 or anti-GST antibodies were used for western blotting. (D) Id2 or Id3 does not bind to AKT1 in Kasumi-1 cells. Immunoprecipitation was performed using anti-Id2 or Id3 antibody, and IgG was used as control. (E) The C terminus of Id1 is required for its interaction with AKT1. Also, Id1 lacking its C terminus does not increase the level of phosphorylated AKT1 in 293T cells. (F) AKT pathway regulators expression in Kasumi-1 cells treated with Id1 inhibitor (15 µM) was examined by doing western blot analysis. (G) In vivo luciferase imaging showed that constitutively activated AKT1 (myristoylated AKT1) can promote leukemia progression in recipient mice transplanted with AE9aId1Δ/Δ cells. (H) Constitutively activated AKT1 (myristoylated [myris] AKT1) promotes leukemia progression in mice transplanted with AE9aId1Δ/Δ cells (n = 10, ***P < .01).