The PI3K-Akt pathway regulates Notch-responsiveness in primary human CD4+ T-cells. (A) 24-well tissue culture plates were coated with anti-human IgG4 and anti-mouse IgG2a capture antibodies at 1 μg/ml in PBS, and incubated at 4°C, overnight. Appropriate stimuli added: Delta1-Fc or hIgG4 as control, both at 20 μg/ml in PBS and anti-CD3 (Hit3a, IgG2a) or mIgG as control, both at 1 μg/ml in PBS, and incubated at 37°C, 3 h. Plates were washed with PBS and positively selected hCD4+ T-cells were seeded at 2 × 106 cells/well, in 1 ml of media to a total of 1 × 107 cells/stimulation. Cells were stimulated overnight ± 10 μM DAPT or 10 μM LY294002 and 2 μg/ml anti-CD28 where applicable. Cells were pooled, RNA prepared and cDNA synthesised as described in Methods. Relative Hes1 expression was determined by Q-PCR. Data shown represents triplicate assays of cDNA prepared from pooled cells, and is representative of two individual experiments performed using CD4+ T-cells prepared from different donors.(B) Human primary CD4+ T-cells were nucleofected with 4 μg p10xCBF-luc, 0.5 μg pTK-RL and 1 μg of either pN1-IC or empty pcDNA3.1, and 2 μg of either gagAkt or kinase-dead (KD) Akt construct. CBF1-reporter activity is presented as mean normalised luminescence ± s.d. Data representative of two repeat experiments from different blood donors.