Western Blot.

B16-F10 or B16.DKO target cells were used to stimulate effector cells. Confluent layers of target cells were established in six-well plates, the medium was removed, and the cells were washed two times with PBS. Effector cells were collected, washed twice, and then incubated in serum-free medium for 2 h before stimulation. Effector cells (5 × 10⁶ in a volume of 1 mL serum-free medium) were gently added to wells containing target cells and incubated at 37 °C for 2, 10, or 30 min. Effector cells were gently collected to avoid contamination with adherent target cells, centrifuged and pelleted, washed with ice-cold PBS, then lysed in RIPA buffer (EMD Millipore) supplemented with Phosphatase Inhibitor Mixture (Roche) and Complete Protease Inhibitor Mixture (Roche). Lysates were cleared of insoluble materials by centrifugation. Protein concentration was measured using the bicinchoninic acid assay assay. Ten micrograms of total protein was resuspended in a reduced sample buffer and was electrophoresed on a 4–12% Bis-Tris gel. For subcellular fractionation, nuclear and cytoplasmic fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) per the manufacturer’s instructions. Briefly, 5 × 10⁶ cells were incubated in 50 μL of cytoplasmic extraction buffer (Thermo Scientific) above ice for 3 min; then the preparation was spun at 20,800 × g for 4 min to isolate cytoplasmic proteins. Nuclear proteins were isolated by adding 50 μL of nuclear extraction buffer (Thermo Scientific) to the pellets. Equal amounts of protein were loaded in each lane, were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen), and then were transferred onto a nitrocellulose membrane. Western blot analyses were performed with specific antibody for pAKT Ser473 [catalog no. 4048; Cell Signaling Technology (CST)], AKT (no. 9722; CST), p-FOXO1 (Thr24)/FOXO3a (Thr32)/FOXO4 (Thr28) (no. 2599; CST), FOXO1 (no. 2880; CST), FOXO3 (no 2497; CST), pSGK1 Ser78 (no. 5599; CST), SGK1 (no. 12103; CST), pGSK-3α/β Ser21/9 (no. 8566; CST), GSK-3β (no. 12456; CST). Blots were visualized using a chemiluminescent substrate (Pierce ECL Plus; Thermo).