ERK signaling in T-LGL is dependent on PI3K activity. (A) T-LGL PBMCs (5 × 10⁷ cells/mL) were untreated (control) or stimulated with IL-2 (20 ng/mL), LY294002 (50 μM), or U0126 (10 μM) for 15 minutes at 37°C. Lysates were prepared by immediate addition of 2 × SDS buffer. After 10% SDS-PAGE and transfer, nitrocellulose membranes were sequentially analyzed for phospho-AKT (S473) and phospho-ERK. (B) T-LGL PBMCs (5 × 10⁷ cells/mL) were untreated or stimulated with IL-2 (20 ng/mL) in the presence or absence of LY294002 (50 μM) for the indicated times at 37°C. Lysates were prepared by immediate addition of 2 × SDS buffer. After 10% SDS-PAGE and transfer, nitrocellulose membranes were sequentially analyzed for phospho-AKT (S473) and phospho-ERK. (C) T-LGL PBMCs (5 × 10⁷ cells/mL) were untreated or with LY294002 (50 μM) or U0126 (10 μM) for 15 minutes at 37°C. Lysates were prepared by immediate addition of 2 × SDS buffer. Samples were subjected to SDS-PAGE and Western blotting for phospho-AKT (S473). For all immunoblots, GAPDH or total AKT were probed to indicate consistency of protein loading between lanes on a given gel. (D) Normal PBMCs and IL-2–activated T cells (T_AC) were incubated with LY294002 (50 μM) or U0126 (10 μM) for 15 minutes at 37°C, or with 1 μg/mL OKT3 for 1 minute prior to lysis in 2 × SDS sample buffer. Proteins were separated on an SDS-PAGE gel as in the previous panels, transferred to nitrocellulose, and immunoblotted for phosphorylated ERK.