Anti pp1α

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).

Cell extracts were prepared using lysis buffer [25 mM Tris-HCl at pH 7.5, 15 mM EDTA at pH 8, 50 mM NaF, 0.6 M sucrose, 15 mM 2-mercaptoethanol, 15 mM Na₄P₂O₇, 1 mM PMSF, and a complete Mini-EDTA free protease inhibitor mixture (Roche Diagnostics, Barcelona, Spain)]. Cells were lysed by repeated passage through 24Gx5/8” needle and centrifuged at 13,000xg 10 min. Thirty micrograms of total protein from the soluble fraction of cell lysates were analyzed by SDS-PAGE and Western blotting using appropriated antibodies: anti-FLAG, anti-HA and anti-actin (Sigma-Aldrich, Madrid, Spain); anti-tubulin, anti-LexA and anti-PP1α (Santa Cruz Biotechnology, Barcelona, Spain); anti-GS (rabbit monoclonal antibody against the C-term of muscular glycogen synthase) and anti-GP (mouse monoclonal against the muscular isoform of the glycogen phosphorylase) (Abcam, Cambridge Science Park, UK); anti-14-3-3ε (Abgent, San Diego, USA) and anti-GFP (ImmunoK, AMS Biotechnology LTD.). Secondary antibodies were from Santa Cruz Biotechnology (Barcelona, Spain). Immunoblots were analyzed by using ECLprime reagent (GE Healthcare, Barcelona, Spain) and chemiluminescence was detected using a Fujifilm LAS- 4000 Lite imager.