Anti β actin af5003

The tissue distribution and relative expression of On-LECT2 in healthy tilapia at the protein level was assessed via Western blotting. Total protein from muscle, skin, brain, head kidney, liver and spleen tissues were isolated using a protein extraction kit (Solarbio, Beijing, China). Each sample containing 20 μg of total protein was loaded on 12% SDS-PAGE and transferred to a PVDF membrane (Millipore). The samples were blocked with QuickBlock™ Blocking Buffer for Western Blot (Beyotime, Shanghai, China) overnight at 4°C and then incubated with anti-LECT2 (PAF541Mu01, Cloud-Clone Corp, Wuhan, China) and anti-β-actin (AF5003, Beyotime, Shanghai, China) as primary antibodies. The solutions were diluted at a ratio of 1:1000 in QuickBlock™ Primary Antibody Dilution Buffer for Western Blot (Beyotime, Shanghai, China) for 2 h at room temperature. Afterwards, the membranes were washed three times in TBST and incubated with HRP-labelled goat anti-rabbit IgG (H+L) (Beyotime, Shanghai, China) at room temperature for 1 h. Antigen–antibody complexes were detected via the enhanced chemiluminescence method (P0018S, Beyotime, Shanghai, China).

Western blotting analysis was performed as detailed previously.^{32 (link)} Antibodies used in this study include anti-YARS2 (ab228957, at a dilution of 1:1000), anti-MT-ND1 (ab181848, at a dilution of 1:2000), anti-MT-ND5 (ab230509, at a dilution of 1:1000), anti-MT-CO2 (ab79393, at a dilution of 1:5000), and anti-TOM20 (ab186735, at a dilution of 1:2000) from Abcam, anti-MT-ND6 (PA5-43532, at a dilution of 1:1000) and anti-MT-ND4 (PA5-97298, at a dilution of 1:1000) from ThermoFisher, anti- MT-CYTB (55090-1-AP, at a dilution of 1:1000) from Proteintech, anti-β-actin (AF5003, at a dilution of 1:2000) from Beyotime Institute of Biotechnology, and HRP-conjugated anti-rabbit secondary antibody (A0208, at a dilution of 1:1000) from Beyotime Institute of Biotechnology. Immunoreactive proteins were visualized using a chemiluminescent immunodetection system (ChemiDoc XRS). Image J was employed to analyze the grayscale values obtained by western blotting.