(A) Schematic of the strategy for generating the Pten^S398A allele, showing the Pten gene with numbered exons (top), the targeting vector (middle), and the recombined locus after excision of the selection cassette (bottom). (B) Western blot, probed with the indicated antibodies, of lysates from Lin^– bone marrow cells from Pten^+/+ and Pten^S398A/S398A (SA/SA) mice, stimulated or not for 15 minutes with 50 ng/mL SCF. The same membrane was simultaneously probed with 4 antibodies using 2 LI-COR fluorescence channels (channel 1: AKT, PTEN; channel 2: pAKT [S473], TUBULIN). See complete unedited blots in the supplemental material. (C) Immunofluorescence microscopy images of cultured HSCs sorted from Pten^+/+ and Pten^S398A/S398A mice, exposed or not to 1 Gy irradiation 4 hours prior to cell fixation. Scale bar: 5 μm. WGA, wheat germ agglutinin. (D) Flow cytometry plots depicting the gating strategy to identify the indicated cell populations in Pten^+/+ (top) and Pten^S398A/S398A (bottom) mice. The parent populations are indicated on top of the plots, and the antibodies used are indicated on the axes. Right: Quantification of the indicated cell populations in Pten^+/+ and Pten^S398A/S398A littermates. Each symbol represents an individual mouse. (E) Diagram representing the experimental setup, and plots showing the proportion of circulating total blood cells, B220⁺ cells, CD3⁺ cells, CD11b⁺ cells, and Gr1⁺ cells expressing the CD45.2 marker in the peripheral blood of lethally irradiated mice transplanted with Pten^+/+ or Pten^S398A/S398A CD45.2⁺ bone marrow cells mixed 1:1 with wild-type CD45.1⁺ competitors. Data are combined from 2 independent experiments in which transplanted cells were pooled from 3 donor animals per genotype; “n” indicates the number of transplanted mice. In all panels, mean and SEM are shown. *P < 0.05; **P < 0.01; ***P < 0.001; assessed by unpaired t test (D) or 2-way ANOVA with Sidak’s multiple-comparison test (E).