Functional evaluation of patient 1 lymphocytes. Activated peripheral blood mononuclear cells (PBMCs) from normal volunteers (NL), a patient with an inactivating FAS mutation (FAS mut), a patient with a gain-of-function somatic NRAS mutation (NRAS mut), and from patient 1 (KRAS mut) were treated for 18 hours with anti-Fas (Apo1.3) antibody (A) or cultured in media without IL-2 for the indicated periods of time (B). (C) Analysis by BIM immunoblotting under IL-2 rich “+” (100 IU/mL) or low “-”(1 IU/mL) conditions in PBMCs from a normal control (NL), a patient with a FAS mutation (FASm), a patient with an NRAS mutation (NRASm), and patient 1 (KRASm). β-actin is a loading control. (D) Activated lymphocytes from a normal control (NL) and patient 1 (KRASm) were cultured in media without IL-2 and treated with dimethyl sulfoxide (DMSO), PD98059 (PD; 20μM), or LY294002 (LY; 10μM) for the indicated periods of time. Apoptosis was measured daily by flow cytometry. (E) Activated PBMCs from normal volunteers (NLs), a patient with an inactivating FAS mutation (FAS mut), a patient with a gain-of-function NRAS mutation (NRAS mut), and from patient 1 (KRAS mut) were cultured in media the indicated concentrations of IL-2, and total cell counts were determined at baseline and 72 hours later; (F) p27^kip1 expression was interrogated by immunoblotting in PBMCs (top) under IL-2 rich “+” (100 IU/mL) or low “-” (1 IU/mL) conditions and also in Jurkat T-cell lines (bottom) transfected with 1 or 6 μg of plasmids encoding GFP-only (negative control), or wild-type, G13C, or G12V (positive control) KRAS. Error bars represent SEs. Data shown are representative of 2 (A-E) independent experiments.