Anti clu

Protein extraction and western blotting were performed as previously described[14 (link)]. Cell lysates were extracted with the T-PER tissue protein extraction reagent (Pierce, Rockford, IL) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. After blocking for nonspecific binding, the membranes were incubated with anti-CLU (1:200 dilution; Santa Cruz Biotechnology), MMP13 (1:200 dilution; Santa Cruz Biotechnology), p-Akt (1:1000 dilution; Cell Signaling Technology), Akt (1:1000 dilution; Cell Signaling Technology), EIF3I (1:800 dilution; Abcam, Hong Kong, China) or GAPDH (1:5000 dilution; Kang-Chen, Shanghai, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using chemiluminescence detection.

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)).