Anti drp1 ab184247

Total cell lysate was prepared from neuronal cells with radioimmunoprecipitation assay buffer (RIPA) supplemented with protease inhibitor (Biochem, PA, United States). Cell concentrations were quantitated by BCA assay and were denatured with SDS-based sample buffer. In Western blot, equal amounts (30 μg) of protein were loaded and separated by SDS-PAGE and then transferred onto the PVDF membrane. The blot was first blocked with 5% non-fat milk and then incubated with primary antibody overnight at 4°C. Antibodies for cleaved caspase 3 (1:1,000; Cell Signaling Technology, MA, United States) and total caspase 3 (1:1,000; Cell Signaling Technology, MA, United States), anti-Drp1 (ab184247), and anti-Fis1 (ab156865) were purchased from Abcam, and anti-March 5 (19168) was purchased from Cell Signaling Technology. Next, the blots were washed with TBST and then incubated with secondary antibodies conjugated with HRP at room temperature for 1 h. Signals were collected by adding ECL solution and were captured by the FluorChem FC2 System (Cell Biosciences, Inc., Santa Clara, CA, United States), and images were analyzed using ImageJ software (NIH, United States).

Anti-ATPB (ATP synthase subunit β) pAb/mAb (ab128743/ab5432), anti-p62 (ab56416), anti-MFN1 (ab57602), anti-MFN2 (ab56889), anti-PINK1 (ab23707), anti-mitochondria [113-1] mAb (ab92824) and anti-DRP1 (ab184247), were purchased from Abcam (Cambridge, MA, USA), anti-ATG5 pAb (NB110-53818) from Novus Biologicals (USA), anti-OPA1 (612606) from BD (USA), anti-Gp78 pAb (16675-1-AP) and anti-ULK1 (20986-1-AP) from Proteintech (USA) and anti-LC3B pAb (2775S) from Cell Signaling. Anti-β-Actin mAb (A5441), tissue culture grade DMSO (D2650), Bafilomycin A1 (Cat# C1988) and CCCP (C2759) were purchased from Sigma-Aldrich (USA). mRFP‑GFP tandem fluorescent‑tagged LC3 (tfLC3) was a gift from Tamotsu Yoshimori (Addgene plasmid # 21074; www.addgene.org/21074) [54 (link)]. mKeima-Red-Mito-7 plasmid was a gift from Michael Davidson (Addgene plasmid #56018; www.addgene.org/56018). mCherry DRP-1 plasmid was a gift from Gia Voeltz (Addgene plasmid #49152 (https://www.addgene.org/49152/). MitoTracker Deep Red FM (M22426), Live Cell Imaging Solution (A14291DJ), Glucose Solution (A2494001), MitoSOX Red (M36008) and MEM Non-Essential Amino Acids Solution (11140050) were purchased from ThermoFisher (USA). MitoView 633 (#70055) was purchased from Biotium (USA).