Supersignal hrp chemiluminescent substrate

The total protein of oviductal tissue was extracted using the proteinase inhibitor-containing lysis buffer. The 4% SurePAGE gel (GenScript, Nanjing, China) was used to separate the equivalent amounts of protein. The separated proteins were placed onto a PVDF membrane (Pall, Mexico) after electrophoresis, and then blocked with sealing solution (Tiangen, Beijing, China). The blocked membrane was incubated overnight at 4 °C. These primary antibodies were anti-synoviolin 1 (SYVN1, 1:2500; Proteintech, Chicago, IL, USA), anti-N1-specific pseudouridine methyltransferase (EMG1, 1:1500; Proteintech, anti-heat shock protein family A (Hsp70) member 8 (HSPA8, 1:4000; Proteintech), and anti-pescadillo ribosomal biogenesis factor 1 (PES1, 1:6000; Proteintech), anti-GAPDH, while horseradish peroxidase (HRP)-conjugated affinipure Goat anti-rabbit IgG (H+L, 1:2000, Proteintech) was used as the secondary antibodies. According to the manufacturer’s instructions, Western blots were visualized on the Odyssey CLX imaging system (Li-COR) (Bio-Rad, USA) with a Supersignal HRP chemiluminescent substrate (Beyotime, Jiangsu, China). The band densities were digitally measured using ImageJ software (NIH).

Goat ovarian tissues were incubated on ice for 30 min in 1.5 mL centrifuge tubes containing lysis buffer, and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected and stored at −80 °C. The protein concentration was measured with a BCA assay kit (Solarbio, Beijing, China), and then 30 μg of protein was mixed with protein loading buffer (Solarbio, Beijing, China) and boiled at 99 °C for 10 min. The protein samples were then subjected to gel electrophoresis at 100 V on a 10% Bis-Tris gel. The proteins were transferred onto a nitrocellulose membrane and blocked at 4 °C overnight. Anti-PPP2R5C and anti-SLC39A5 were purchased from Proteintech company (Proteintech, Wuhan, China). All primary antibodies were diluted at 1:10,000 with primary antibody diluent (P0256-500 mL, Bain-marie, China) and washed three times with TBST before being washed with HRP-conjugated anti-rabbit IgG (Proteintech, Wuhan, China) with secondary antibody dilution (P0258-500 mL, Baymax, China) diluted to 1:5000 used to incubate the membrane for 2 h at room temperature. Western blots were visualized on an Odyssey CLX imaging system (Li-COR) (Bio-Rad, Hercules, CA, USA) with Supersignal HRP chemiluminescent substrate (Beyotime, Beijing, China) according to the manufacturer’s instructions.