PI3K-AKT pathway activity in T-LGL cells. (A) Lysates of purified CD8 T cells or PBMCs before and after adherent cell depletion, as indicated, from healthy donors and patients with T-LGL were prepared by direct addition of 2 × SDS sample buffer after a brief 15-minute incubation at 37°C and separated on a 10% SDS-PAGE gel. Following transfer to nitrocellulose membrane, samples were sequentially analyzed for phosphorylated AKT (S473) and total AKT protein via immunoblotting. Cell equivalents (3 × 105) were loaded in all lanes. (B) PBMCs from 4 patients with T-LGL were incubated at 37°C for 15 minutes in the presence or absence of LY294002 (50 μM), followed by immediate addition of 2 × SDS sample buffer. Proteins were separated on an SDS-PAGE gel as in panel A, transferred to nitrocellulose, and immunoblotted for phosphorylated GSK-3β (S9). GAPDH levels were probed to indicate consistency of protein loading.
