Mitofusin 2

For protein content analysis, 30 μg of total protein from each plantaris muscle was separated by SDS-PAGE, transferred onto a PVDF membrane, and immunoblotted as previously described [33 (link)]. The following antibodies (Cell Signaling, Danvers, MA) were used: Ulk1 (1:1000), phospho-Ulk1 (1:1000), Drp1 (1:1000), beclin-1 (1:1000), SQSTM1/p62 (1:1000), Bnip3 (1:1000), LC3B (1:1000), and mitofusin-2 (1:1000). Membranes were analyzed and quantified using Bio-Rad Laboratories Image Lab software (Hercules, CA).

Mdivi-1 was obtained from APExBIO Technology LLC, TX, USA, Unitaxel (Paclitaxel) was from Hikma, Egypt (Concentration 300 mg paclitaxel/50 ml), and cisplatin was purchased from Mylan, Viatris, PA, USA. Drp1-specific siRNA was obtained from Santa Cruz Biotech Inc, TX, USA, for siRNA transfection, Lipofectamine 2000 in OptiMem reduced serum media was from ThermoFisher Scientific, USA, and Wortmannin (Wort) was purchased from Toronto Research Chemicals (Canada). Monoclonal antibodies including Drp1, Mitofusin-1, and Mitofusin-2 were from Cell Signaling Technology, USA. MTT was from Sigma and cell culture media were from Lunza, Pharma Biotech. Breast cancer cells (MDA MB-231) were kindly provided by the Department of Cancer Biology, NCI, Cairo, Egypt.

10% tissue homogenates from the frontal cortex and hippocampus were prepared in lysis buffer, centrifuged at 13,500 g for 30 minutes at 4°C. Protein concentrations of each sample were determined by the BCA protein assay kit with BSA standards. 60 μg of total proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated for 1 hour with 5% dry skim milk in TBST buffer at room temperature to block nonspecific binding. Membranes were then incubated with primary antibodies against caspase-3 at a dilution of 1:1000 (the rabbit anti-mouse cleaved caspase-3 mAb, Cell Signaling Technology, USA), Bcl-2 at 1:1000 (the rabbit anti-mouse Bcl-2 mAb, Cell Signaling Technology, USA), Mfn2 at 1:1000 (the rabbit anti-mouse mitofusin-2 mAb, Cell Signaling Technology, USA), Drp1 at 1:1000 (the rabbit anti-mouse Drp1 mAb, Cell Signaling Technology, USA), β-actin at 1:2000 (ZS-Bio, China) overnight at 4°C. Subsequently, membranes were incubated with alkaline-phosphatase-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium (SIGMA, USA). The band density was scanned and analyzed using the Odyssey Two-Color Infrared Imaging System (LI-COR, USA).

Cell lysates prepared with NuPAGE^TM LDS sample buffer (4X) (Invitrogen) were electrophoresed on NuPAGE 4–12% Bis-Tris Gel (Life Technologies), and proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes with Trans-Blot Turbo PVDF Transfer kit (Bio-Rad). PVDF membranes were blocked with 10% skim milk (Sigma-Aldrich) in TBS, followed by incubation with indicated primary and secondary antibodies (GE healthcare). The specific proteins were detected with the Pierce ECL Western Blotting Substrate (Thermo Scientific). ImageJ was used for quantifications of bands on Western blots.
In this study, the following mouse monoclonal primary antibodies (mAbs) were used: V5-tag (#46-0705) (Invitrogen); OPA1 (#612607), Drp1 (#611113), and Myc-tag (#551101) (BD Biosciences); GAPDH (#sc-32233) (Santa Cruz); and Mitofusin 1 (#ab57602) (Abcam). Rabbit polyclonal antibodies (pAbs) were MIEF2 (#HPA042334), Fis1 (#HPA017430) (Atlas Antibodies); MIEF1 [25 (link)]; Mitofusin 2 (#9482S), PARP (#9542) (Cell Signaling); and VDAC1 (#ab15895) (Abcam). The following secondary antibodies were used: The peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies (GE Healthcare) for immunoblotting and the DyLight 488- and 649-conjugated anti-mouse and anti-rabbit IgG antibodies (Vector Laboratories) for immunofluorescence.

Cells were lysed in RIPA buffer (#9806S; Cell Signaling) with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (#78444, Thermo Fisher Scientific) unless indicated otherwise. Antibodies were used according to the manufacturer's instructions to detect the following antigens: AMPK (Cell signaling, #5831), p‐AMPK (Thr172) (Cell signaling, #2535), pULK1 (Ser555, Cell signaling, #5869), ULK1 (Cell signaling, #6439), p62 (Cell signaling, #39749), β‐actin (Santa Cruz, #sc‐1616), DRP1 (Cell signaling, #8570), p‐DRP‐1 (S616, Cell signaling, #3455), Beclin1 (Cell signaling, #3738), mitofusin 2 (Cell signaling, #9482), total OXHOS rodent cocktail (Abcam, ab110413), SOD‐1 (Cell signaling, #2770), SOD‐2 (Enzo Life Sciences, #ADI‐SOD‐110‐F), γ‐H2AX (Santa Cruz, #sc‐517348), and GAPDH (Abclonal, #AC027).