Anti p drp 1 ser616

Bafilomycin A1 (B1793), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (C2759), propidium iodide (P4170), oligomycin (O4876), 2-[2-[4(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) (C2920), antimycin A (AA) (A8674), and rotenone (R8875) were purchased from Sigma-Aldrich. SYTO-13 (S7575) was purchased from Thermo Fisher Scientific. The Masson’s Trichrome staining kit was obtained from RAL Diagnostic (RAL-361350-0000). The DeadEnd^TM Fluorometric Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System was purchased from Promega (G3250). Anti-cleaved-caspase 3 (Asp175) (9664), anti-LC3B (2775), anti-VDAC1 (4661), anti-ATG7 (2631), anti-Beclin 1 (3728), anti-GRP75 (2816), and anti-p-Drp-1 (Ser616) (3455) antibodies were from Cell Signaling Technology. Anti-β-actin (A2228) and anti-SQSTM1/P62 (P0067) were from Sigma-Aldrich. Anti-TOMM 40 antibody (H-300) was from Santa Cruz Biotechnologies. Anti-MOMA 2 antibody (MAB1825) was from Merck Millipore, anti-Parkin (ab15954) and anti-PINK1 (ab74487) were from Abcam. Anti-PGC-1α antibody (NBP1-04676) was from NOVUS Biologicals. Anti-TFEB antibody (A303-673A) was from Bethyl Laboratories. Smooth muscle actin rabbit polyclonal antibody (RB-9010-P) was from Thermo Fisher Scientific. Secondary antibodies conjugated to horseradish peroxidase (HRP) were from Cell Signaling Technology.

The hippocampus or cells were lysed in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors and homogenized for 2 min. Protein concentrations were then measured using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, P0010). The loading volume was calculated according to a protein content per well of 40 μg; the experimental protocol was consistent with that of a previous study.²⁷ After blocking with 5% Albumin Bovine V (Solarbio, A8020) in Tris‐buffered saline containing 0.1% Tween 20 for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: anti‐Drp1, anti‐Fis1, anti‐OPA1, anti‐Mfn1, anti‐Mfn2 (Santa Cruz, CA, USA), anti‐p‐Drp1 (Ser637), anti‐p‐Drp1 (Ser616), anti‐cytochrome oxidase subunit IV (COXIV; Cell Signaling Technology), anti‐sequestosome 1, (p62; Cell Signaling Technology), anti‐microtubule‐associated protein 1 light chain 3β, (LC3B, Cell Signaling Technology and Abcam), and anti‐superoxide dismutase 2, (SOD2, Proteintech). Subsequent incubation was performed using goat anti‐rabbit/mouse secondary antibodies for 1 h at room temperature. The membranes were then imaged via enhanced chemiluminescence using an Odyssey infrared imaging system (LI‐COR Biosciences).

For immunofluorescence analysis, pOBs were induced to mineralization and treated on glass in the conditions described above. Then, cells were fixed in 4% PFA for 15 min at RT, permeabilized with 0.2% Triton™ X-100 (Sigma-Aldrich) in PBS, and blocked with PBS 5% FBS for 1 h at RT. The primary antibodies anti-p-DRP1[Ser616] (Cell Signaling Technologies, Danvers, MA, USA) were diluted 1:250 in blocking buffer and incubated for 2 h at RT. The secondary antibody conjugated with Alexa Fluor 488 (donkey anti-rabbit IgG, Invitrogen Thermo Fisher Scientific) was diluted in blocking buffer 1:300 at a concentration of 0.0067 mg/mL, and incubated for 45 min at RT. Nuclei were visualized with DAPI (Sigma Aldrich). Images were acquired with fluorescence microscope LEICA DMi8 equipped with Leica Application Suite LAS X Imaging Software. ImageJ software (NIH Image, Bethesda, MD, USA) was used to analyze fluorescence intensity and data were represented as fluorescence intensity/cell area versus G condition.