Anti chop

CD4⁺ T cells lysate was prepared by lysing in RIPA buffer containing protease inhibitors. Protein concentrations were estimated (BCA protein assay kit). After that, the protein samples were loaded onto each lane by SDS-PAGE for electrophoresis and transferred to 0.45 μM PVDF membrane (Millipore, MA, USA). Next, PVDF membranes were blocked with 5% skimmed milk in TBS-Tween for 60 min, followed by incubation with antibody at 4°C for 12-16 h. The following primary antibodies are as follows: anti-mTOR (Cat#AF6308, dilution rate 1 : 1000), anti-P-mTOR (Cat#AF3308, 1 : 1000), anti-P-p70s6k (Cat#AF3228, 1 : 1000), anti-p70s6k (Cat#AF6226, 1 : 1000), anti-4EBP (Cat#AF6432, 1 : 1000), anti-caspase-3 (Cat#AF6311, 1 : 1000), anti-Bcl-2 (Cat#AF6139, 1 : 1000), anti-Bax (Cat#AF0120, 1 : 1000), anti-GRP78 (Cat#AF5366, 1 : 1000), anti-CIRP (Cat#DF2643, 1 : 1000), anti-CHOP (Cat#DF6025, 1 : 1000), anti-CIRP (Cat#AF5366, 1 : 1000), and anti-actin-β (Cat#AF7018, 1 : 3000) were purchased from Affinity Biosciences (Jiangsu, China). Anti-P-4EBP (anti-eIF4EBP1, ab27792) was from Abcam (CA, USA). After washing with TBS-Tween 3 times, the membranes were incubated with a goat anti-rabbit IgG antibody (1 : 5000, Affinity Biosciences) at 25°C for 1 h, and the chemiluminescence signals were detected using an electrochemiluminescence detection system. Band densities were quantified by the ImageJ software.

Total protein was extracted from cells with RIPA lysis buffer (Beyotime, China) after incubation for 48 h. After quantification of the protein concentration with a BCA protein assay kit (Beyotime, China), proteins in the samples (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% fat-free milk and incubated with diluted primary rabbit polyclonal antibodies, including anti-GHSR-1a (1:1000, Affinity, USA), anti-PERK (1:1000, Affinity, USA), anti-p-PERK (1:2000, Affinity, USA), anti-AFT4 (1:1000, Affinity, USA), anti-CHOP (1:1000, Affinity, USA), and anti-GAPDH (1:1000, Hangzhou Xianzhi Biology Co., Ltd., China) antibodies, overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Boster, China) as the secondary antibody for 2 h at room temperature. The bands were visualized by ECL and quantified using Bandscan software 5.0 (Glyko Inc., CA, USA).