Bicinchoninic acid assay

Cells were collected and lysed in RIPA lysis buffer (Yeasen, catalog #20101ES60) containing a protease inhibitor cocktail (Yeasen, catalog #20104ES08). Protein concentration was quantified by bicinchoninic acid assay (Thermal Fisher). Equal amounts of soluble protein were loaded and separated on a 10% SDS-PAGE, followed by transfer to nitrocellulose and then immunoblotting using primary antibodies, including p53 (CST, catalog #32532), p21 (abcam, catalog #ab109199), c-Myc (CST, catalog #13987 T), c-Maf (abcam, catalog #ab77071), p-STAT3 (CST, catalog #9145), t-STAT3 (CST, catalog #9139), PD-L1 (R&D, catalog #AF1019), Caspase 3 (CST, catalog #9665S), ZAP70 (CST, catalog #3165S), MDM2 (BD, catalog #556353), and β-actin (CST, catalog #3700S). HRP-conjugated secondary antibodies (Yeasen, catalog #33101ES60, catalog #33201ES60) were used at 1:5000 dilution.

The cell line was characterized in vitro to test for the expression of human PSA by Western blotting and IF. For Western blotting, the total protein from the cloned cells was extracted and its concentration was determined using a bicinchoninic acid assay (Thermo Scientific). Protein samples were run on 12% SDS-PAGE gels and were transferred to polyvinyldene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were incubated overnight using rabbit anti-human PSA antibody (Cell Signaling Technology, Danvers, USA) as a primary antibody at 1000× dilution. Next, the membranes were washed and immediately incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies.
For IF, pre-blocking clone cells were incubated with the primary antibodies overnight in a humidified chamber at 4°C. All specimens were then washed with PBS and immediately incubated with fluorochrome-conjugated secondary antibody [Alexa Fluor 488 goat anti-rabbit IgG (green), Life Technologies] diluted in antibody dilution buffer for 1 h at room temperature in a dark environment. Slides were covered with mounting medium with DAPI (blue, Vector Laboratories, Inc., Burlingame, CA, USA). Negative controls were performed on RM9 cell lines. Confocal laser scanning microscopy was performed with Zeiss LSM 780 (Carl Zeiss, Oberkochen, Germany) using 40× oil objective lens, as indicated.

Cerebral cortex samples from five adult and five aged dairy cattle were treated with lysis buffer (SDS) 5% (Sigma-Aldrich) and ammonium bicarbonate 50 mM (BioRad), and were subjected to 3 cycles of high-frequency sound wave sonication via a probe inserted in the sample, which creates an area of low pressure to favor the disruption of tissue. Following incubation at 4°C for 45 min on a bench spinning shaker, samples were centrifugated (30 min at 13,000 rpm). Supernatants were collected, and protein extracts were quantified by bicinchoninic acid assay (Thermo Fisher Scientific). Of each protein sample, 50 μg was digested by trypsin onto S-Trap filters according to the manufacturer’s protocol (ProtiFi, Huntington, NY) (Palinski et al., 2021 (link)). Peptide mixtures were dried in a SpeedVac system (Thermo Fisher Scientific).

The sperm samples collected from three mature spermiating males were centrifuged individually at 300× g at 4 °C for 30 min followed by 10 min at 13,000× g at 4 °C. The supernatant (seminal plasma) was carefully collected and stored at −80 °C. The pellets (spermatozoa) were collected and suspended in protein extraction buffer (8 M urea, 2 M thiourea, 4% CHAPS, 10% w/v isopropanol, 0.1% w/v Triton X-100, 100 mM dithiothreitol) containing HALT protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The suspended samples were vortexed continuously for 1 h, and subsequently centrifuged for 10 min at 13,000× g at 20 °C. The supernatant containing proteins was collected separately and stored at −80 °C. Bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration in the samples of seminal plasma and spermatozoa, using the Infinite M200 photometer (Tecan, Männedorf, Switzerland).

The collected cells were washed with PBS and then lysed with RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Total protein amount was measured with a bicinchoninic acid assay (Thermo Fisher Scientific), and 30 µg total lysate per sample was subjected to SDS-PAGE followed by immunodetection with the following primary antibodies: Snail (CST; 3879), N-cadherin (CST; 13116),Vimentin (CST; 5741), E-cadherin (CST;3195), GAPDH (CST;5174), Twist (CST;46702), and Smad2/3 Antibody Sampler Kit (CST; 12747). For detection, the corresponding HRP-linked secondary antibody (CST) and enhanced chemiluminescence (Pierce, USA) were added.