Ripa lysis buffer

Total protein was extracted from cells, which were lysed by radioimmunoprecipitation assay (RIPA) Lysis Buffer (BB-3209, BestBio Co., Ltd., Shanghai, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride (PVDF) blotting membrane. After being blocked with a blocking solution for 1 h, the membrane was incubated with primary rabbit monoclonal antibodies, including anti-CD9 (1:2000, ab92726), anti-CD63 (1:1000, ab213090), anti-CD81 (1:1000, ab109201) and anti-FAM13A (1:500, ab122440) antibodies, overnight at 4 °C. All of the above antibodies were purchased from Abcam (Cambridge, MA, USA). The next day, the membrane was incubated with a horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:1000, Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) for 1 h at 37 °C with shaking. The internal reference was GAPDH, and each experiment was repeated 3 times.

Tumor tissues were removed 24 h after the last administration to analyze PLK-1 expression in vivo. Tumor fragments (100 mg) were processed for total mRNA or protein extraction followed by qRT–PCR and western blot assays, respectively. The extracted mRNA samples were standardized to the same absorbance value of 260 nm and the expression of the PLK-1 mRNA was detected using qRT–PCR as described above. The selected tumor tissues were homogenized in 1 mL of RIPA lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT, 100 mM AEBSF, 100 μM aprotinin, 5 mM bestatin, 1.5 mM E64, 2 mM leupeptin, 1.5 mM pepstatin A, cyclosporin A, sodium fluoride, beta-glycerophosphoric acid disodium salt, sodium orthovanadate, disodium molybdate dihydrate, and sodium pyrophosphate) (BestBio, China) supplemented with PMSF (1 mM) to evaluate PLK-1 protein levels. The lysates were incubated on ice for a total of 30 min and vortexed every 5 min. After purification and quantification, the protein levels were determined using western blot analysis as described above.

RAW264.7 cell and BMDM lysates were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (BestBio, Shanghai, China, Cat#: BB-3201) for total protein isolation and a Nuclear Protein Extraction Kit (Solarbio, Beijing, China, Cat#: R0050) for nuclear protein isolation according to the protocol recommended by the manufacturers. Western blotting was performed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% non-fat milk, the membranes were incubated with primary antibodies against collagen I (1:50, Abcam, Cat#: ab270993), NR3C2 (1:1,000, Proteintech, Cat#: 21854-1-AP), TGF-β1 (1:1,000, Abcam, Cat#: ab215715), α-SMA (1:1,000, ABclonal, Cat#: A17910), and vimentin (1:1,000, Abcam, Cat#: ab8978) overnight at 4°C. The next day, the blots were incubated with fluorescein-conjugated secondary antibodies at 1:10,000–1:20,000 for 1 h at room temperature and scanned with a Dual Color Infrared Laser Imaging Scanner (Odyssey, LICOR, Lincoln, NE, USA). Protein expression was measured with ImageJ by quantifying the density of the target total protein relative to GAPDH (1:1,000, Proteintech, Cat#: 60004-1-lg), beta-tubulin (1:1,000, Affinity, Cat#: T0023), or the target nucleoprotein relative to proliferating cell nuclear antigen (PCNA) (1:1,000, Proteintech, Cat#: 10205-2-AP).