Bicinchoninic acid protein detection kit

After abdominal anesthesia (1% sodium pentobarbital, 120 mg/kg), 2 mL of carotid artery blood was extracted and centrifuged. Rat serum testosterone was detected by enzyme-linked immunosorbent assay (ELISA) according to the instructions of the Rat Serum Testosterone Assay Kit (Lengton Bioscience Co, Ltd). The rat penile tissue was divided into 3 sections after removal of the foreskin, glans, and urethral spongiosum. These 3 sections were then examined by transmission electron microscopy, Western blot detection, and nitric oxide assays, respectively.¹² (link)^,15 (link) Total protein was isolated from rat penile tissue by centrifugation and collected. Protein concentration was determined with a colorimetric method via a bicinchoninic acid protein detection kit (Beyotime Biotechnology Co, Ltd). The concentration of nitric oxide was measured with a colorimetric method (Elabscience Biotechnology Co, Ltd).¹³ (link)^,14 (link)

Total proteins in the colonic tissues (serious ulcer, atypical hyperplasia, or tumor tissues) were separated using radioimmunoprecipitation assay buffer (ApplyGen., Beijing, China). The concentrations of the above-­isolated proteins were determined with a bicinchoninic acid Protein Detection kit (Beyotime Biotech., Shanghai, China) as instructed by the manufacturer. Proteins were separated with 10% sodium dodecyl sulphate-­polyacrylamide gel electrophoresis (Amersham Biosciences, Piscataway, NJ, USA) and electro-­transferred onto the polyvinylidene fluoride (PVDF) membrane (DuPont, Wilmington, Del, USA). Then, the PVDF membrane was washed with the Tris-buffered saline tween 20, blocked with 5% non-fat milk at room temperature for 1 hour, and incubated with mouse STAT3 antibody (Cat. No. MAB1799, RD Systems, Minneapolis, Minn, USA), phospho-STAT3 antibody (Cat. No. AF4607, RD Systems), mouse cyclin D1 antibody (Cat. No. AF4196, RD Systems), mouse suppressor of cytokine signaling 3 (SOCS3) antibody (Cat. No. MAB5696, RD Systems), and mouse glyceraldehyde-3-phosphate dehydrogenase antibody (Cat. No. A01622, GenScript., Nanjing, China) at 4°C overnight. Subsequently, PVDF membrane was washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cat. No. 12-349, Sigma-Aldrich, St. Louis, Miss, USA) or HRP-conjugated goat anti-rabbit IgG (Cat. No. 12-348, Sigma-Aldrich), or at room temperature for 1 hour. Eventually, the PVDF membrane was imaged using enhanced chemiluminescence kit (Thermo Scientific Pierce, Rockford, Ill, USA) as per the manufacturer’s protocol.

Murine BMSCs were obtained from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China) and cultured in MSC growth medium (Lonza Group Ltd., Basel, Switzerland) at 37 °C with 5% CO₂. BMSCs of the 4th–6th generation were used for subsequent experiments. BMSCs were identified according to the method reported in the previous literature¹⁷ . The extraction of BMMSC derived extracellular vesicles (B-EVs) was carried out according to the previous reports^{7 (link),18 (link)}. BMSCs were seeded in six-well plates with MSC growth medium at a density of 1.03 × 10⁵/well. After 1 day of culture, the cells were washed with phosphate-buffered saline (PBS) three times, and the medium was replaced by EVs-free medium for 48 h. Then, 2 mL conditioned medium (CM) was collected and centrifuged at 300 × g and 4 °C for 10 min to remove cells, then centrifuged at 2000 × g for 10 min to remove cell debris, and then centrifuged at 10,000 × g for 35 min. Next, the supernatant was filtered by 0.22 μm filtration membrane (Steritop™ Millipore, Burlington, MA, USA) and centrifuged at 180,000 × g for 70 min. The precipitate was resuspended in PBS and centrifuged at 180,000 × g for 70 min. The precipitate was finally resuspended with 100 μL PBS. Bicinchoninic acid protein detection kit (Beyotime Biotechnology Co., Ltd, Shanghai, China) was used for quantitative analysis of EV protein in subsequent experiments. At the same time, an inhibitor of EV secretion GW4869 (20 μg/mL; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was added to the medium without EV serum. After 48 h of culture, the supernatant was taken as the methods of EV isolation as the negative control (NC). The B-EVs were then identified using nanoparticle tracking analysis (NTA, Malvern, Chester County, PA, USA), transmission electron microscope (TEM) and western blot analysis. The diameter of murine B-EVs was analyzed by using NTA. The selected concentration of the samples was 1–9 × 10⁸ cells/mL, and the appropriate gray background was selected by the software and the movement of the particles was recorded. The distribution of concentration and variation of the substitute sample was drawn.