Odyssey fluorescence imager

T-cells from co-culture experiments (1 × 10⁵ T-cells seeded) were harvested with one part of 1× PBS and one part of Novex^® buffer (Thermo Fisher Scientific) containing 3% dl-dithiothreitol (Sigma-Aldrich). Samples were run on 10% SDS PAGE gels and then blotted on an Amersham protan nitrocellulose transfer membrane (Sigma-Aldrich). Phospho-protein phosphatase 2A (pPP2A) (1:7,000, clone E155; Abcam, Cambridge, UK), protein phosphatase 2A (PP2A) (1:1,000), pAkt (Ser 473, 1:2,000, clone D9E), pAkt (Thr 308, 1:1,000, clone C31E5E), Akt (1:1,000, clone C67E7, all Cell signaling technology, Danvers, MA, USA), and vinculin (clone hVIN-1, 1:40,000 Sigma-Aldrich) protein expressions were determined by immunoblotting of CD3⁺ T-cells at intervals up to 96 h after allogeneic stimulation with DCs. Western blots for pPP2A and pAKt (Ser473 and Thr308) were developed by chemiluminescence imaging using the Super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific) and chemiluminescent detection films (Sigma-Aldrich). Western blots for PP2A, Akt, and vinculin were developed by fluorescence imaging using Goat-anti-mouse IgG Dylight 800 and Goat-anti-rabbit IgG Dylight 800 (both Thermo Scientific) on an Odyssey Fluorescence imager (Licor, Licoln, NE, USA). Western blots were analyzed by densitometry using the ImageJ software.

Erythrocyte ghosts were prepared as described previously (20 (link)) from rabbit blood (Pel-Freez). Briefly, 5 ml of packed RBC were washed twice in 0.9% NaCl and incubated in 90 ml of lysis buffer (5 mM Na-phosphate buffer, 1 mM EDTA, pH 7.4) overnight at 4°C under constant stirring. Ghosts were obtained after centrifugation at 15,000 × g for 20 min, washed 3 times with lysis buffer, washed one time in phosphate-buffered saline (PBS), and resuspended in PBS to an OD₆₀₀ of 0.2. Heptamers were formed by incubating 5 μl ghosts with purified AT proteins (0.5 μg) and PBS in a final volume of 21 μl for 45 min at 37°C. Samples were then solubilized in 7 μl of Bolt LDS sample buffer (Invitrogen) and incubated for 5 min at room temperature, and 13 μl was run on 4 to 12% Bolt morpholinepropanesulfonic acid (MOPS) SDS gel (Life Technologies). The separated proteins were transferred to nitrocellulose membrane in Bolt transfer buffer (Invitrogen) with 10% methanol overnight at 15 V, blocked with Odyssey blocking buffer (LI-COR) for 1 h, and probed with rabbit anti-AT IgG (2 μg/ml) for 2 h at room temperature. The AT bands were detected after 1 h of incubation with IRDye 680RD-conjugated donkey anti-rabbit IgG (LI-COR) by an Odyssey fluorescence imager (LI-COR). Band intensities were calculated with Odyssey Image Studio Lite software.