Anti cirp

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.