Tanon 5200

RIPA buffer (Thermo Fisher Scientific) was applied to lyze CRC cells and the total protein in the lysates were then quantified using a BCA Protein Assay kit (Thermo Fisher Scientific). Cellular proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, proteins were transferred onto polyvinylidene difluoride membranes. Subsequently, after being blocked, membranes were incubated with primary antibodies against β-actin (1:1000 dilution; Abcam), HIF1α (1:1,000 dilution; Proteintech, Wuhan, China) and CPT1C (1:1000 dilution; Proteintech) overnight at 4°C, followed by incubation with the corresponding HRP-conjugated secondary antibody. Finally, ECL reagents (Thermo Fisher Scientific) were used for blot visualization and quantification on a BioImaging System (Tanon 5200, Shanghai, China). β-actin was used as the loading control.

Total protein was extracted from cells or left ventricular tissues. The whole operation was carried out on ice. The collected cells or the ground tissues were lysed with RIPA lysis buffer, which added protease inhibitors. Lysates were centrifuged for 15 min at 12,000 rpm under 4℃. The supernatants were used for Western blotting after determining its protein concentration by Bradford assay (Yeasen, China). The protein was separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking, PVDF membranes were incubated with different primary antibodies overnight at 4℃ or 2 h at 25℃.
Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca_V1.2 α_1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus). The secondary antibodies used were HRP-conjugated goat anti-mouse (0.02 µg/mL, SA00001-1, Proteintech) or anti-rabbit IgG (0.02 µg/mL, SA00001-2, Proteintech), or rabbit anti-goat IgG (A21030, Abbkine). We used an imaging system (Tanon 5200, China) to visualize the blot with the enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific).

The reproductive organ tissues of boars from various age groups, the proteins present in adult boar semen, and the proteins from RAW264.7 cells were isolated using a 12% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After 1 h of blocking with 5% skim milk powder at room temperature, PVDF membranes were washed with TBST (137 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) and incubated with primary antibodies overnight at 4 °C. Anti-CRISP3, Anti-CRISP2 antibody (1:2000, SAB2501636, Sigma, Marlborough, MA, USA), IL-1α, and IL-6 (1:1000, Abcam, Waltham, MA, USA) and control antibodies β-actin and GAPDH (1:2000, TransGen, Beijing, China) were employed. After washing the membranes, Goat Anti-Rabbit IgG (H + L) Horseradish Peroxidase (HRP)or Goat Anti-Mouse IgG (H + L) HRP (1:2000, Thermo Fisher Scientific, Waltham, MA, USA) were incubated for 1 h. Automatic imaging equipment (Tanon5200, Shanghai, China) was used to image the membranes after washing and incubating them with chemiluminescence solution in a dark room for 2 min. The experiment was repeated at least three times. The Image J software (https://imagej.nih.gov/ij/index.html, accessed on 23 January 2024) was utilized for the comparative analysis of protein expression levels.