Western blot analysis of Akt, phosphorylated Akt and IκB-α
PBMC were incubated with anti-CD3 (10 μg/ml) in the presence or absence of LY294002 (20 μM). After incubation for 1 hour, whole cell lysates were prepared from about 107 cells by homogenization in the lysis buffer, and centrifuged at 14,000 r.p.m. (19,000 g) for 15 min. Protein concentrations in the supernatants were determined with the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, membrane was preincubated with 0.1% skimmed milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 hours, then primary antibodies against Akt, phosphorylated Akt and IκB-α (Cell Signaling Technology Inc., Beverly, MA, USA), diluted 1:1000 in 5% BSA/TBS-T, were added and incubated overnight at 4°C. After washing four times with TBS-T, horseradish peroxidase-conjugated secondary antibodies were added and allowed to incubate for 1 hour at room temperature. After TBS-T washing, hybridized bands were detected with the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).