Tbst buffer

We carried out western blot to verify the expression of the lysis protein E. Briefly, the overnight cultured E. coli BL21(DE3) cells were transferred to fresh LB medium at a 1% ratio. We added 1 mM IPTG to induce the protein E during the logarithmic growth period and we continued the cultures for 15, 30, and 60 min. Next, we collected the samples by centrifugation at 12,000 rpm for 2 min and resuspended the cell pellets in lysis buffer. The cell mixture was lysed in a sonicator for 5 min and then boiled for 10 min. Then, we loaded 10 µL protein samples on a 16% Tricine gel for SDS-PAGE and transferred the separated proteins onto a nitrocellulose membrane (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) in a Bio-Rad Transblot Turbo system at 80 V for 1 h. After incubation with the 5% (w/v) non-fat milk powder in TBST buffer (Sangon Biotech, Shanghai, China) for 1 h at room temperature, the membrane was washed twice with TBST buffer for 15 min each time. Next, we incubated the membrane with anti-Strep-Tag II monoclonal antibody (Abbkine, Wuhan, China) at 4 °C overnight, and the next day, with HRP-conjugated goat anti-mouse IgG H&L (Abcam, Cambridge, UK) for 1 h at room temperature. Finally, the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize the immunoreactive protein.

The isolated liver samples were homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Beyotime Biotechnology, China) to extract total protein, which concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology). The aliquots of protein samples (20μg) were separated by 10% SDS-PAGE gel and then transferred to activated polyvinylidene fluoride membranes (Merck Millipore, Germany). After being blocked with 5% skimmed milk in TBST buffer (Sangon Biotech, China), the membranes were incubated with the primary antibody overnight in a refrigerator at 4°C and then with secondary antibody for 2h. The immune complexes were visualized with a Beyo ECL Plus kit (Beyotime Biotechnology) and quantified using ImageJ software (Bethesda, United States). The specific primary antibodies are anti-FoxO1 (ET1608-25, HuaBio, China), anti-phospho-FoxO1 (#9461, Cell Signaling Technology, United States) anti-Akt1 (#4691, Cell Signaling Technology, United States), anti-phospho-Akt1 (#4060, Cell Signaling Technology, United States), anti-Sirt1 (sc-74,465, Santa Cruz Biotechnology), anti-Nrf2 (ab62352, Abcam, China) and anti-β-tubuliin (#2146, Cell Signaling Technology, United States) antibodies. The secondary antibodies are anti-mouse antibody (Solarbio, China) and anti-rabbit antibody (SongonBiotech, China).

Sertoli cells were sowed at 2 × 10⁶ cells/well in medium dishes and processed according to experimental requirements. Sertoli cells were digested in a centrifuge tube, rinsed once with PBS, and an appropriate protein lysis buffer (RIPA, Beyotime) was added (dependent on cell volume). The supernatant was collected by centrifugation at 4 °C. The protein concentration was measured using a BCA kit (Beyotime), and the protein was boiled 5 times in protein loading buffer for 10 min. A total of 60 μg of protein was isolated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was incubated with a blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h and then incubated with a primary antibody at 4 °C overnight. The membrane was washed 5 times with TBST buffer (Sangon Biotech, Shanghai, China), incubated with the second antibody for 1 h at room temperature, and then washed 5 times with TBST. The protein bands were analyzed on a chemical isotope imaging system (CLiNX Scientific Instruments, Shanghai, China). The antibody information is provided in Table 4.