Anti phospho drp1 s616

The lumbar region of the spinal cord was collected from WT and SOD1 G93A mice and sonicated in a lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10% Glycerol, 1% Triton-X100 and 1 mM EDTA). After protein quantification using the BCA method, 30 µg of protein was loaded per well and separated by SDS-PAGE. The loaded protein was transferred to a PVDF membrane, and this membrane was incubated in a blocking solution (3% BSA/1 ×TBST) for 1 h. The membrane was incubated with primary antibody diluted 1:1000 with blocking solution overnight at 4 °C, then washed three times in 1xTBST and incubated with secondary antibody diluted 1:5000 with 5% Skim Milk/1xTBST. The antibodies used were anti-Drp1 (BD, Cat. 611113), anti-phospho Drp1 S616 (Cell signaling, Cat. 3455), anti-Fis1 (Abcam, Cat. Ab96764), anti-PP1α (Santa Cruz, Cat. sc-443), anti-phospho-PP1α (Cell Signaling, Cat. #2581), PP1β (Santa Cruz, Cat. sc-373782), PP1γ (Santa Cruz, Cat. sc-6109), and anti β-actin (Sigma, Cat. A5441). The secondary antibodies used were anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase. The membrane was washed three times in 1xTBST, and the signals expressed by the protein were visualized using an ECL kit (Thermo, Cat. 32106).