CLL cells purified from spleens of 8-month-old Eμ-TCL1 mice express reduced amounts of AKT. (A) PBMCs isolated from 8-month-old wild-type mice were stained with CD19-APC-Cy7, IgM-Alexa568, CD5-APC, B220–Alexa 488, and DAPI. Live PBMCs were analyzed for CD5⁺/B220⁺ CLL cells on gated CD19⁺/IgM⁺ B-cell populations. Data are representative of 3 independent experiments. (B) PBMCs isolated from Eμ-TCL1 mice of different age groups were stained with CD19-APC-Cy7, IgM–Alexa 568, CD5-APC, B220–Alexa 488, and DAPI. Live PBMCs were analyzed for CD5⁺/B220⁺ CLL cells on gated CD19⁺/IgM⁺ B-cell populations. Data are representative of more than 4 independent experiments. (C) CD5⁺/B220⁺ CLL were plotted against PBMCs or CD19⁺/IgM⁺ B cells. Each dot represents an individual mouse, and horizontal bars indicate means of at least 4 experiments. (D) IgM⁺ cells purified from spleens of Eμ-TCL1 mice were analyzed for the presence of CD5⁺/B220⁺ CLL cells. Data are representative of 3 independent experiments. (E) Lysates from CD5-/B220⁺ B cells of 6-week-old wild-type (WT) and Eμ-TCL1 mice (lanes 1 and 2), from CD5-/B220⁺ B cells of 8-month-old wild-type mice (lane 3), and from CD5⁺/B220⁺ CLL cells of 8-month-old Eμ-TCL1 mice (lane 4) were immunoblotted for TCL1, AKT, p97, and actin. Results shown in each immunoblot are representative of 3 independent experiments. For each experiment, CD5⁻/B220⁺ B cells, CD5⁻/B220⁺ Eμ-TCL1 B cells, and CD5⁺/B220⁺ CLL cells were purified and pooled from at least 2 spleens of mice with indicated genotypes.