Tim 3

Equal amounts of protein from total-cell lysates were separated by 12% SDS-PAGE (Beyotime) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked at room temperature (20–25°C) for 2.5 h in 5% non-fat dry milk in TBS-T. Membranes were incubated with gentle rocking 1.5 h at room temperature with primary antibodies for Tim-3 (1:2,000, Proteintech, China), Arg-I (1:1,000, Proteintech, China), iNOS (1:1,000, Abcam, UK), PTEN (1:600, Proteintech, China), PI3K (1:500, Proteintech, China), AKT (1:500, SAB, USA), pAKT (1:500, SAB, USA), SOCS1 (1:600, Proteintech, China), C/EBPβ (1:750, SAB, USA), TNF-α (1:2,000, Proteintech, China), IL-10 (1:5,000, Proteintech, China); GAPDH (1:40,000, Proteintech, China) was used as a loading control. Membranes were washed 5 times with TBS-T for 10 min each time and then incubated with the appropriate secondary Ab for 2.5 h at room temperature. The immune complex was visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were determined using the Image J software (Rawak Software, Inc., Germany).

CD3^-CD56⁺ dNK cells from the three groups were incubated for 36 h before harvesting. Equal amounts of protein from total-cell lysates were separated by 12% SDS-PAGE (Beyotime) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked at room temperature for 2.5 h in 7% nonfat dry milk in TBS-T buffer. Membranes were incubated with gentle rocking 1.5 h at room temperature with primary antibodies for Tim-3 (1/2000, Proteintech), GranzymeB (1/2000, Abcam), Perforin (1/1000, Proteintech), GranzymeA (1/600, Proteintech), PI3K (1/500, Proteintech), AKT (1/500, SAB), pAKT (1/500, SAB), STAT1 (1/500, Proteintech), STAT3 (1/600, Proteintech), pSTAT1 (1/500, Abcam), pSTAT3 (1/600, Abcam), IL-10 (1/500, Abcam), IFN-γ (1/500, Proteintech) and GAPDH (1/40000, Proteintech) as a loading control. Membranes were washed with TBS-T 5 times for 10 min each, then incubated with the appropriate secondary antibody for 2 h at room temperature. Then immune complex was visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were determined by Image J software (Rawak Software, Inc., Germany).