Nucleic acid and protein microanalyzer

Total protein samples were extracted using RIPA lysis buffer. A nucleic acid and protein microanalyzer (Molecular Devices, United States) was used to determine protein concentrations. Western blotting was performed according to a published protocol (Wu et al., 2022 (link)). Primary antibodies were incubated overnight at 4°C as follows: anti-iNOS (Proteintech, 22226-1-AP, 1:5000), anti-PD-L1 (Proteintech, 66248-1-Ig, 1:3000), anti-Arg-1 (Proteintech, 16001-1-AP, 1:2000), anti-GADPH (Proteintech, 60004-1-Ig, 1:10000), anti-CD9 (Proteintech, 20597-1-AP, 1:2000), anti-CD63 (Proteintech, 25682-1-AP, 1:2000), anti-TSG101 (Proteintech, 28283-1-AP, 1:3000), anti-JAK2 (Cell Signaling Technology, 3230S, 1:2000), anti-STAT3 (Proteintech, 10253-2-AP, 1:5000), anti-p-STAT3 (Cell Signaling Technology, 9145S, 1:1000), and anti-IL-6 (Cell Signaling Technology, 12912S, 1:1000). The HRP-conjugated secondary antibody (ZSGB-BIO, ZB-2305; ZB-2301) was incubated for 1 h at room temperature. The signal was detected using the ECL method (Beyotime, China, P0018FS). Band density was quantified using ImageJ software (NIH, Bethesda, MD, United States) for the grayscale intensity of each protein.

Protein samples were prepared using a RIPA lysis buffer. The concentration of the protein was analyzed using a nucleic acid and protein microanalyzer (Molecular Devices, San Jose, CA, USA). SDS–PAGE was performed to separate the proteins, which were then electrotransferred onto a PVDF membrane. The membrane was incubated with antibodies at 4 °C overnight. Next, the membrane was washed and incubated with an HRP-conjugated secondary antibody (Beyotime, BeiJing, China). Finally, signals were detected by the ECL method (Beyotime, BeiJing, China) and analyzed using ImageJ software.

Tumor tissues and HepG2 cells were lysed in an RIPA buffer solution containing PMSF and protein inhibitors (Roche, Germany). A nucleic acid and protein microanalyzer (Molecular Devices, United States) was used to determine the protein concentration. The proteins were separated using 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk for 2 h and incubated with primary antibodies at 4°C overnight. The membranes were washed with PBST, and the protein bands were detected by the ECL and were exposed to the TANON gel imager (Shanghai, China). The results were analyzed by ImageJ software. Because the molecular weights of the target proteins were the same, the primary and secondary antibodies were removed using a stripping buffer (P0025N, Beyotime Biotechnology, China) and then the membranes were washed with PBST and finally blocked and incubated with the antibodies for subsequent experiments.