Nf κb p65

The protein extraction and quantification were applied according to our previous literature^{13 (link)}. The protein concentration was determined by the bicinchoninic acid (Beyotime Institute of Biotechnology, China) method. The protein samples were separated by SDS-PAGE (Beyotime Institute of Biotechnology, China) electrophoresis. The film was transferred under the condition of voltage 100 V, current 120 mA, 90–120 min. It was then sealed in a sealant (5% skim milk) for 2 h. TTBS was washed off the sealing fluid, and the primary antibody was diluted with primary antibody diluen: ZAK (1:500; abcam, USA), p-NFκB-p65 (1:1000; Thermo Scientific, Beijing, China), NFκB-p65 (1 µg/ml; Thermo Scientific, Beijing, China), ZO-1 (1:300; Thermo Scientific, Beijing, China), occludin (1:200; Thermo Scientific, Beijing, China), and claudin-5 (1:500; Thermo Scientific, Beijing, China) in a certain proportion and sealed with a film at 4 °C overnight. After washing TTBS for three times, add the corresponding secondary antibody and incubate at room temperature for 2 h. After TTBS washing for three times, ECL (Beyotime Institute of Biotechnology, China) was used to glow, take photos, and quantity one software was used for quantitative analysis.

IF staining was conducted as previously described^{22 (link)}. The cultured cells were fixed at 4 °C in ice-cold methanol for 10 min, washed three times in PBS, and then permeabilized in 0.1% Triton X-100/PBS for 10 min at room temperature. Nonspecific binding was blocked with 0.5% Tween-20/PBS containing 3% BSA for 30 min. The cells were incubated with primary antibodies (#436700, NF-κB P65, Thermo Fisher; #4053, PKM2, CST) at 4 °C overnight. The cells were then incubated with secondary antibodies (#A-11078, Alexa Fluor 488 goat anti-rabbit IgG, #31660, rhodamine goat anti-mouse IgG, Thermo Fisher) for 1 h at room temperature. The incubated cells were washed with PBS, and Hoechst 33342 (#H1399, Thermo Fisher) was used to visualize the nuclei. The cells were incubated at room temperature for 5 min, washed three times with PBS, and observed under a confocal microscope (Olympus).

The lysates of cell culture and mouse lungs were prepared in a lysis buffer in the presence of protease/phosphatase inhibitors, and BCA protein quantification was then performed. Denatured protein samples were loaded on 10–15% SDS-PAGE gels and transferred to PVDF membranes. After blocking the membranes with 5% skimmed milk in TBST, the membranes were incubated with the following primary antibodies: phosphorylated (p)-IκBα (cat. no. 9246), p-NF-κB p65 (3033), p-c-Jun (32740), c-Jun (9165), p-c-Fos (5348), and c-Fos (2250) (all 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA); IκBα (cat. no. MA5-15132) and β-actin (MA5-15739) (both 1:1000; Invitrogen; Thermo Fisher Scientific, Inc., Rockford, IL, USA); NF-κB p65 (cat. no. sc-8008), iNOS (sc-651), COX-2 (sc-1747), and Lamin A/C (sc-376248) (all 1:1000; Santa Cruz Biotechnology, Inc., CA, USA). Then, the membranes were maintained with the corresponding secondary antibodies, washed with PBS, and exposed to ECL solution to visualize each band.

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.

RIPA lysis buffer and Bradford Protein Assay Kit were used for protein determination from brain tissues (Bio Basic Inc., Markham, Ontario, Canada). The separated proteins were loaded and incubated on the PVDF membrane with primary antibodies for Beclin 1 (# PA5-20172), p-ERK (#44-680G), p-mTOR (#44-1125G), p-PI₃K (# PA5-99367), ERK ( #13–6200), mTOR (# AHO1232), LC₃ A (#PA5-23180), LC₃ B (#PA1-16930), NF‐κB (p65) (# 51–0500), and β-actin (# MA5-15739) (Thermo Fisher Scientific Inc., Waltham. MA, USA) at 4°C. Following washing, blots were incubated with peroxidase‐labeled anti‐rabbit secondary antibodies at 37°C for 1 h. protein Bands intensity was detected using the chemiluminescent substrate (ClarityTM Western ECL substrate—Bio-Rad, TNC, USA), and a CCD camera-based imager. Analysing the images was done using Chemi Doc MP Imager and then normalised by β-actin.