Clone c67e7

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.

Antibodies against human Akt (rabbit monoclonal; mAb, clone C67E7, working dilution: 1/300), FoxO1 (rabbit mAb, clone C29H4, working dilution: 1/200), and PRAS40 (rabbit mAb, clone D23C7, working dilution: 1/500) were from Cell Signaling Technology (Danvers, MA, USA). The antibody against human Akt phosphorylated at T308 (phosphoAkt-T308; mouse mAb, working dilution: 1/25) was purchased from OriGene (Rockville, MD, USA), whereas the one recognizing the phosphorylated at S473 (phosphoAkt-S473) molecule (rabbit mAb, clone EP2109Y, working dilution: 1/150) was from Abcam (Cambridge, UK). The antibodies to phosphorylated FoxO1 (phosphoFoxO1-S319, rabbit polyclonal, working dilution: 1/1000), PRAS40 (phosphoPRAS40-T246, rabbit polyclonal, working dilution: 1/200), and CD21 (rabbit mAb, EP3093, working dilution: 1/500) were also from Abcam. The antibody to CD3 (rabbit mAb, working dilution: 1/500) was from Cell-Marque (Rocklin, CA, USA), whereas antibodies to CD20 (mouse mAb L26, working dilution: 1/200) and cytokeratin (mouse mAb, clone MNF116, working dilution: 1/400) were from Dako (Glostrup, Denmark). The EnVision system (Dako) recognizing mouse and rabbit antibodies was employed as a second antibody and development system.

Cells were lysed in cold RIPA lysis buffer and Halt™ Protease and Phosphatase Inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) for 20 min on ice. The cell lysates were clarified by centrifugation at 10,000×g for 20 min. Proteins (10–25 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked in TBS-T buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) non-fat milk at room temperature for 1 h and then probed at 4 °C overnight with antibodies to detect AKT (pan) (clone C67E7, 1:1000), phospho-AKT (Ser473) (clone D9E, 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (clone D13.14.4E, 1:1000), p44/42 MAPK (ERK1/2) (clone 137F5, 1:2000) from Cell Signaling Technology (Boston, MA, USA); Gαq (clone ab75825 and ab190082, 1:1000) from Abcam (Cambridge, USA); DYKDDDDK Tag Monoclonal Antibody (FG4R, MA1-91878, 1:1000) from Thermo Scientific (Waltham, MA, USA), and GAPDH (clone 60004-1-Ig, 1:2000) from ProteinTech (Chicago, IL, USA). Detection was carried out with the SuperSignal West Femto Maximum Sensitivity Substrate Trial kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA).