Bca reagent

The collected human spermatozoa samples were denatured individually in 8 M urea/1 M NH₄HCO₃ and went through ultrasonication on ice by Ultrasonic Cell Distribution System. Thereafter, denatured samples were centrifuged at 15,000g for 20 min, and supernatant was collected for protein concentration measurement by BCA reagent (Beyotime). Spermatozoa proteins (1 mg) were reduced by 5 mM dithiothreitol (DTT) at 37 °C for 1 h with gentle shaking, then alkylated by 15 mM iodoacetamide at room temperature (RT) for 30 min in the dark. Subsequently, another 2.5 mM DTT was added and incubated for 10 min at RT. Protein samples were diluted twofold with deionized water and digested by sequencing grade trypsin (protein: enzyme, 100:1, w/w; Promega) at 37 °C for 2 h with gentle shaking as the first digestion. Afterward, samples were diluted fourfold with deionized water and sequencing grade trypsin (protein: enzyme, 100:1, w/w; Promega) was used to digest proteins into peptides again by incubation at 37 °C with gentle shaking overnight, namely the second digestion. The samples were acidified with trifluoroacetic acid (TFA) and centrifuged at 15,000g for 15 min to remove any particulate matter. The digested peptides were desalted with C18 column (Waters) and eluted with 50% acetonitrile (ACN)/0.1% TFA. The peptide concentration was measured by BCA reagent (Beyotime).

RAW264.7 cells and colon tissues were homogenized, and protein levels were quantified using BCA reagent (Beyotime, Shanghai, China). Nuclear and cytoplasmic extractions were performed as instructed by the manufacturer (Beyotime, Shanghai, China). Samples were loaded and electrophoresed in 10–12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk diluted in TBS-T for 2 h followed by incubation with primary antibodies. The following primary antibodies were used in this experiment: iNOS, COX-2, phospho-IKKα/β, IKKα/β, phospho-IκBα, IκBα, phospho-ERK ½, ERK ½, phospho-p38, p38, p65, phospho-c-Jun, c-Jun, c-Fos, β-tubulin, Lamin A/C, phospho-JNK, JNK, IRAK4, and β-actin at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG at 1:2,500 dilution.

Total proteins were extracted from cortical tissues. BCA reagent (Beyotime, Shanghai, China) was used to measure the concentration of extracted proteins. A total of 10 μg protein sample was loaded for polyacrylamide gel electrophoresis (10% gel for TrkB, 12% gel for BDNF), and were transferred to polyvinylidene fluoride membrane (Pall Life Science, Port Washington, NY, USA) in a 200 V electrical field for 30 min. The membrane was first blocked for 1 h using 5% defatted milk powder, and then was incubated with rabbit anti-BDNF (1:2000, Abcam, Cambridge, MA, USA), goat anti-TrkB (1:1000, R&D System, Minneapolis, MN, USA) or rabbit anti-tubulin (1:2000, Abcam) primary antibody at 4°C overnight. Excess antibody was then washed by PBST, followed by incubation in horseradish peroxidase (horseradish peroxidase-conjugated goat anti-rabbit IgG (1:8000, ServiceBio, Wuhan, China) or mouse anti-goat IgG (1:10 000, ServiceBio) for 1 h at room temperature. The membrane was developed in ECL chromogenic substrates, and was imaged by an automatic protein imaging system (Bio-Rad, Hercules, CA, USA). Image J (NIH, Bethesda, MD, USA) software was used to measure optical density values of each band, which was normalized to that of tubulin. Each experiment was performed in triplicates.