T-cell and thymocyte signaling in T-cell–specific PTEN-null and PTEN–phosphatase-dead mice. (A) Western blot analysis of PI3K/Akt signaling in total thymocytes of 4- to 5-week-old WT, PTEN-ΔT, and PTEN^C124S/ΔT mice, representative of >6 experiments. Crosslinking of anti-CD3 (3 μg, clone 2C11) was achieved using anti-hamster IgG, with or without the addition of 1 μg soluble anti-CD28. (B) Phospho-flow analysis of PI3K/Akt signaling in total thymocytes, gated on either CD4 single-positive (CD4SP) or CD4/CD8 double-positive (DP) cells, representative of >4 experiments. The same activation conditions as western blot experiments, with anti-CD28, were used for phospho-flow. (C) Western blot analysis of PI3K/Akt signaling in purified CD4⁺ premalignant T cells from spleen and lymph nodes of 4- to 5-week-old WT, PTEN-ΔT, and PTEN^C124S/ΔT mice, representative of >6 experiments. Crosslinking of anti-CD3 (3 μg, clone 2C11) was achieved using anti-hamster IgG, and 1 μg soluble anti-CD28 was added on the addition of anti-CD3. (D) Quantitative analysis of western blots of peripheral CD4⁺ T cells stimulated for 15′ with anti-CD3 ^+/− anti-CD28. WT stimulated with anti-CD3 alone (without anti-CD28) is set to a relative unit of 1, to which all genotypes and stimulation with anti-CD28 are compared, for each phosphoprotein/total protein. Results are for 3 independent experiments.