PI3K activity in T-LGL is dependent on the activation of a Src family kinase. (A) PBMCs from 3 patients with T-LGL were incubated at 37°C for 15 minutes in the presence or absence of the SFK inhibitor PP2 (10 μM), followed by immediate addition of 2 × SDS sample buffer. Cell equivalents (3 × 10⁵)/lane were run on a 10% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted for phosphorylated AKT (S473) and total AKT. (B) CD8⁺ T cells isolated from a healthy donor and a patient with T-LGL via RosetteSep were resuspended in RPMI at 5 × 10⁷ cells/mL and incubated at 37°C for 15 minutes, followed by immediate addition of 2 × SDS sample buffer and immunoblotted with an antibody specific for active SFKs. (C) CD8⁺ T cells isolated from a patient with T-LGL via RosetteSep were resuspended in RPMI at 5 × 10⁷ cells/mL and incubated at 37°C for 15 minutes in the presence or absence of LY294002 (50 μM) or PP2 (10 μM), as indicated. Lysates prepared as for panel B were immunoblotted for phospho-SFK. Total AKT and GAPDH protein levels are shown as loading controls.