Western blotting was performed as previously described (Sen et al., 2013 (link); Sen et al., 2015 (link)). In short, protein samples from adult flies were extracted in a 1X SDS sample buffer. samples were run on 4%–15% TGX gels (Cat # 4561086, Bio-rad Laboratories, Hercules, CA) and transferred on to a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA, United States) using a Trans-blot Semi-dry apparatus (Cat # 1073940, Bio-rad Laboratories, Hercules, CA). After blocking, blots were probed against appropriate primary and secondary antibodies. anti-Clu (1:15,000) (Cox and Spradling 2009 (link)), anti-TOM20 (1:2000, Santa Cruz Biotechnology, Dallas, TX), anti-ATP5A (1:100,000, Abcam, Cambridge, United Kingdom). The following antibodies were kindly provided by Dr. Edward Owusu-Ansah from Columbia University Medical Center, NY: anti-ND-ASHI (1:3,000), anti-ND-SGDH (1:3,000), anti-ND-17 (1:3,000), anti-ND-17.2 (1:3,000) and anti-UQCR-C2 (1:3,000) (Murari et al., 2020 (link)).
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