Goat anti mouse igg dylight 800

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.

Whole cell protein lysates were prepared from HUVEC using CelLytic reagent (Sigma). Immunoblotting of cell lysates was performed according to standard conditions. Immunoblots were labelled with the following primary antibodies: anti-Akt (11E7) (4685, 1:1,000, Cell Signaling Technology), anti-phospho (S473)-Akt (9271, 1:1,000, Cell Signaling Technology), anti-ERG (sc353, 1:500, Santa Cruz Biotechnology), anti-ERG (ab133264, 1:1,000, Abcam), anti-Dll4 (1:500, R&D systems), anti-GAPDH (MAB374, 1:10,000, Millipore), anti-Jag1 (sc-6011, 1:1,000, Santa Cruz Biotechnology), anti-NICD/cleaved Notch1 (Val1744) (2421, 1:500, Cell Signaling), anti-Tie2 (D9D10) (7473, 1:1,000, Cell Signaling). Primary antibodies were detected using fluorescently labelled secondary antibodies: goat anti-rabbit IgG DyLight 680 and goat anti-mouse IgG Dylight 800 (Thermo Scientific). Detection and quantification of fluorescence intensity were performed using an Odyssey CLx imaging system (LI-COR Biosciences, Lincoln) and Odyssey 2.1 software. In some instances, HRP-conjugated secondary antibodies were used for chemiluminescence detection and protein levels were quantified by densitometry and normalized against loading controls. See Supplementary Fig. 10 for the uncropped immunoblots.