Pa523170

The immunostaing was performed as described previously^{17 (link)}. Briefly, RAW264.7 cells were fixed in 4% (w/v) paraformaldehyde for 10 min at room temperature, and washed three times with PBS, then permeabilized with 0.3% (w/v) Triton X-100 for 20 min at room temperature, followed by three washes with PBS. After incubation in PBS containing 10% bovine serum albumin for 30 min at room temperature, the slides were incubated with antibody against p-p65-NLS (PA523170, Thermo Scientific Inc., Waltham, MA, USA) overnight at 4 °C and then incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h in the dark. To stain the nuclei, 1 µg/ml of 4′,6-diamidino-2-phenylindole in PBS was added before capturing images with a fluorescence microscope (Olympus CKX41, Tokyo, Japan).

Protein lysates were isolated from cells or tumor sections using RIPA buffer (CST, Danvers, MA, USA). Protein separation was performed using electrophoresis in 10–15% arc-bis gels, and the proteins were transferred onto PVDF using a transfer system (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the appropriate primary antibodies (1:1000) and secondary antibodies (Thermo, A11034, A32723 1:10000), reacted with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA), and incubated for several minutes in a dark room. Antibodies against PD-L1 (Cell Signaling Technology, 60475, 1:1000), NAMPT (Proteintech, MA5-24108, 1:1000), β-actin (Abcam, ab8226, 1:1000), GAPDH (Abcam, ab8245, 1:1000), Ac-tubulin (Abcam, ab24610, 1:1000), α-Tubulin (Abcam, ab7291, 1:1000), Ac-NF-κB p65 (K310) (Thermo, PA5-17264, 1:1000), NF-κB p65 (Thermo, PA5-23170, 1:1000), HIF-1α (Thermo, MA1-516, 1:1000) were purchased from commercial company.