Bicinchoninic acid bca kit

Male C57BL/6 mice and female BALB/c mice were purchased from Shanghai Model Organisms Center Inc. Lipopolysaccharide and TUNEL kits were purchased from Sigma, USA. In total, 10% chloral hydrate was purchased from Zhujiang Hospital of Southern Medical University. Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore, USA. Protein primary antibodies were purchased from cell signaling technology, USA. Protein secondary antibody and bicinchoninic acid (BCA) kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad, USA. EPO (product batch number: 20,100,901, product specification 5000 IU/branch) was purchased from China resources Angde Biotech Pharma Co., Ltd. 4% paraformaldehyde was purchased from Guangzhou Chemical Reagent Factory (GCRF). Paraffin, xylene, and neutral gum were purchased in Beijing Solarbio Science & Technology Co., Ltd. SDS-PAGE gel preparation kit and RIPA lysate were purchased from Wuhan Servicebio Co., Ltd. Malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

Hep‐2 and TU212 cells in logarithmic phase were inoculated at 6 × 10⁵ cells/dish in 100 mm × 20 mm dishes and allowed to grow for 48 hours. After incubation with 0, 10, 30 and 50 μg/mL SM‐BFRE for 24 hours, the cells were collected by centrifugation and lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime) to extract the proteins. The supernatant containing the protein was collected, and the concentration of the protein was measured by the bicinchoninic acid (BCA) kit (Beyotime). After denaturation, the protein was stored in a −80°C freezer until required. Protein extraction from the tumour tissues was performed following the same protocol. Equivalent amounts of the protein (40 µg) were separated by SDS‐PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad). The membranes were blocked by 5% skim milk formulated with tris‐buffered saline with 0.5% Tween 20 (TBST) for 2 hours at room temperature. The membranes were then incubated with the primary antibody at 4°C overnight and incubated with the secondary antibody at room temperature for 1‐2 hours. The HRP ECL system (Bio‐Rad) was used to detect the protein band, and the grey value was calculated by Image Lab software.

Fresh unpasteurized milk (2 liters) was collected from the Weizhu Dairy located in Nanjing, China and stored at 4 °C for 2 hours before the isolation of exosome.
Exosomes were isolated from milk using the differential centrifugation as described previously.^{10,29 (link)} Briefly, milk was centrifuged at 3000 × g for 15 min at 4 °C to remove fat globules, cellular debris and somatic cells. The whey was collected by passing through a cheese cloth and transferred into polycarbonate tubes. In order to remove large particles, it was centrifuged for 60 min at 100 000g in Type 45Ti fixed angle rotor using Optima LE-80K Ultracentrifuge (Beckman Coulter, Brea, California). Supernatant was finally centrifuged at 135 000 × g for 90 min at 4 °C. Then the final supernatant was discarded and the exosome pellet was washed twice using the phosphate-buffered saline (PBS). The exosome pellet was re-suspended in PBS, followed by filtration through a 0.22 μm Steritop filter (Millipore, Darmstadt, Germany). The total protein concentration was determined using the bicinchoninic acid (BCA) kit (Beyotime Biotechnology, China). The concentration of the milk derived exosomes was adjusted to 6 mg ml⁻¹ and stored at −80 °C until further use. The isolated exosomes were characterized as recommended by the International Society of Extra Cellular Vesicles (ISEV).^{30 (link)}

Based on the previous study [21 (link)], 200 μL Radioimmunoprecipitation (RIPA) solution (Beyotime, Shanghai, China), which contains 1x protease inhibitor, was added to crack the crypts and shaken at 4 °C for 20 min to fully crack the crypts. The protein concentration was determined by Bicinchoninic Acid (BCA) kit (Beyotime, Shanghai, China). After adding the protein loading buffer, the protein was denatured at 100 °C for 10 min and stored at −80 °C refrigerator. The protein samples with the calculated concentration were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis. Then, they were gummed, transferred, sealed, and incubated primary antibody overnight at 4 °C. Protein primary antibody information and dilution ratio: BAX (1:1000, Proteintech, Wuhan, China), Caspase9 (1:1000, CST, Boston, MA, USA), PCNA (1:1000, Abcam, Cambridge, UK), Olfm4 (1:1000, CST, Boston, MA, USA), P-YAP (1:1000, Abclonal, Wuhan, China), YAP (1:1000, Abcam, Cambridge, UK), Cyclin D1 (1:1000, CST, Boston, MA, USA). Finally, we incubated secondary antibodies, and the ECL solution was used to reveal the bands. A total of 30 experimental animals C57BL/6 mice were used.

The morphology of Fe₃O₄/ATX and Fe₃O₄/ATX/Transferrin was evaluated using TEM (Tokyo JEOL, Japan). Dynamic light scattering (DLS) (Malvern ZS90, United Kingdom) was used for measuring the hydrodynamic size distribution. The iron concentration of the resulting NPs was analyzed on a UV-vis spectrophotometer (UV-3600, Shimadzu, Japan) by 1, 10-phenanthroline spectrophotometric method. The excitation and emission wavelength were detected on a fluorescence spectrometer (HJY-FL3-211-TCSPC, France). Bicinchoninic acid (BCA) kit (Beyotime Biotechnology, Shanghai, China) was used to evaluate the coupling content of transferrin on NPs by the microplate reader (BioTek ELx808, United States). The standard concentration and non-encapsulated amounts of ATX were measured at OD430 nm by the same microplate reader, calculating the entrapment efficiency of ATX-NPs.
Mean values were reported from three individual experiments.