Western Blotting Procedure for Protein Analysis

Western blotting analysis was carried out as detailed elsewhere (60 (link),62 (link)). Fish were sacrificed after anesthesia and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty micrograms of total proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)]. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was carried out as detailed previously (60 (link)).

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Zhang Q., He X., Yao S., Lin T., Zhang L., Chen D., Chen C., Yang Q., Li F., Zhu Y.M, & Guan M.X. (2021). Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency. Nucleic Acids Research, 49(8), 4689-4704.

Publication 2021

RIPA reagent AffiniPure goat anti-mouse IgG goat anti-rabbit IgG ECL system

Anesthesia Anti igg Antibodies Bis tris Fish Gapdh Goat Membrane Mouse Peroxidase Polyacrylamide gels Polyvinyl Protein Rabbit Ripa Tfam Western blotting analysis

Corresponding Organization : Women's Hospital, School of Medicine, Zhejiang University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of Mto1, Gapdh, Nd1, Sdha, Atp5a, Mtpap, Uqcrc2, Co2, Atp8, Ndufs1, Cox5a, Atp5c, Tfam, Tufm, and Tom20

control variables

None explicitly mentioned

controls

Positive controls: None mentioned
Negative controls: None mentioned

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