Peroxidase affinipure goat anti mouse igg and goat anti rabbit igg

Fish were sacrificed after anesthesia, and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty μg of total cellular proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. After transfer, the membrane was incubated in 1 × PBST (1 × PBS, 1% Tween 20) and 5% nonfat dry milk for 1 h. After blocking, the membrane was incubated with primary antibody overnight at 4°C following with secondary antibody. The hybridized membrane was then exposed to chemiluminescence reagent for 1 min and developed by ChemiScope 3300 mini (CLiNX, Shanghai). The antibodies were obtained from different companies including Mtu1 from Hangzhou HuaAn Biotecnology Co (HuaBio), Gapdh (SAB2701826) from Sigma-Aldrich, Nd1 (ab74257), Nd6 (ab81212), Atp5a (ab188107), Sdhb (ab151684) and Yars2 (ab127542) from Abcam, Cytb (A9762) from ABclonal, Co2 (55070-1-AP), Kars (14951-1-AP), Lars2 (17097-1-AP), Tufm (26730-1-AP), Tfb2m (24411-1-AP) and Tom20 (1802-1-AP) from Proteintech. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (30 (link),42 ).

Western blot analysis was performed as detailed elsewhere.²² (link)^,33 (link) Twenty micrograms of cellular proteins was electrophoresed through 10% Bis-Tris SDS-polyacrylamide gels (Thermo Fisher Scientific) and then transferred to a polyvinylidene difluoride membrane. The primary antibodies used for this experiment were obtained from Abcam (Cambridge, UK), including YARS2 (ab127542), ND1 (ab74257), CO2 (ab110258), TOM20 (ab56783), and total OXPHOS human WB antibody mixture (ab110411), and from Proteintech (Rosemont, IL, USA), including NDUFS1 (12444-1-AP), NDUFA9 (20312-1-AP), NDUFS3 (15066-1-AP), SDHA (14865-1-AP), UQCRC1 (21705-1-AP), COX5A (11448-1-AP), ATP5B (17247-1-AP), and VDAC (55259-1-AP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AB-M-M 001) was obtained from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, China), and light chain 3 (LC3; 4108) from Cell Signaling Technology (Danvers, MA, USA). Peroxidase AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG (The Jackson Laboratory, Bar Harbor, ME, USA) were used as a secondary antibody, and protein signals were detected using the ECL Western Blotting Analysis System (MilliporeSigma, Billerica, MA, USA). Quantification of density in each band was performed as detailed previously.²² (link)^,33 (link)