Fig. 6: ASXL1-MT/BAP1 complex deubiquitinates and stabilizes Akt.

a 293T cells were transfected with FLAG-ASXL1-WT, FLAG-ASXL1-MT, and AKT1 expressing vectors. Total cell lysates were subjected to immunoprecipitation using anti-FLAG antibody followed by immunoblotting. b 293T cells were transfected with AKT1 and BAP1 expressing vectors. Total cell lysates were subjected to immunoprecipitation using anti-AKT antibody followed by immunoblotting. c 293T cells were transfected with AKT1, FLAG-ASXL1-MT, BAP1, and Myc-Ubiquitin expressing vectors. Total cell lysates were subjected to immunoprecipitation using anti-AKT antibody followed by immunoblotting. Relative levels of Myc-Ubiquitin were quantified by densitometry and normalized to total Myc-Ubiquitin levels. d 293T cells were transfected with AKT1, FLAG-ASXL1-WT, FLAG-ASXL1-MT, BAP1, and Myc-Ubiquitin expressing vectors. Total cell lysates were subjected to immunoprecipitation using anti-AKT antibody followed by immunoblotting. Relative levels of Myc-Ubiquitin were quantified by densitometry and normalized to total Myc-Ubiquitin levels. e–g Murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells) were transduced with Cas9 and sgRNA targeting Bap1 (e). Bap1-depleted cSAM cells were then starved of IL-3 for 3 h followed by stimulation with IL-3 (1 ng/mL) for the indicated times (f). Relative levels of phosphorylated Akt were quantified by densitometry and normalized to total Akt levels (g). Similar result was obtained from Bap1-depleted cSAM cells using another sgRNA-targeting BAP1 (sgBAP1 #2). A representative experiment from at least n = 2 independent experiments is shown.