P gcn2

Western blot analysis was performed as previously described42 . DR4 antibody was from Novus (Novus Biologicals, Littleton, CO, USA); DR5 and P62 antibodies were from Abcam (Cambridge, UK); FLIP-L and FLIP-S antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Caspase 3, Caspase 8, Caspase 9, PARP, CHOP, PERK, p-GCN2, GCN2, p-PKR, PKR, HRI, p-eIF2α, eIF2α, ATF4, MAP1LC3B, tubulin, GAPDH and β-actin antibodies were from Cell Signaling Technology (Beverly, MA, USA); The secondary antibodies used were HRP-conjugated anti-mouse IgG and anti-rabbit IgG from sigma (USA).

CD4⁺ T cells were lysed in 50ul of lysis buffer (4.8% SDS, 8% sucrose, 2M urea) containing protease inhibitor cocktail (Calbiochem), for 30 minutes on ice and centrifuged for 15 minutes at 15,000 rpm. Protein concentrations were determined by Quick Start Bradford assay kit (Bio-Rad), according to the manufacturer's instructions. Protein lysates were denatured at 95°C for 5 minutes. Protein samples (25μg) were separated on 10% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) and probed using the following antibodies: pGCN2, ATF4, GAPDH and ß-actin (Cell Signalling, Danvers, MA, USA). Detection of the immunoreactivity bands was performed with the ECL Western Blotting Substrate (Bio-Rad), and chemiluminescence was detected with the ImageQuant imaging system using anti-rabbit or anti-mouse HRP-linked antibody (eBioscience).