Sab3700861

Astrocytes were lysed by RIPA buffer and approximately 50μg of total protein was diluted in sample buffer (Laemmli‐BioRad, USA) containing 20mg/ml of DTT (Sigma‐Aldrich, USA). Proteins were denatured by heating (5min at 95°C), and then separated by electrophoresis in a 10% polyacrylamide gel. Subsequently, the proteins were transferred to a nitrocellulose membrane, and blocked for 1h in 5% milk dissolved in Tris‐Buffered Saline (TBS) containing 0.05% of Tween‐20 (TBS‐T) for further incubation with primary antibodies overnight. After this incubation, the membrane was washed with TBS‐T and incubated for 1h with secondary antibodies conjugated with Horseradish peroxidase (HRP). The molecular mass of the proteins was determined by comparison with the migration pattern of Spectra Multicolor Broad Range Protein Ladder (Thermo, USA). Membranes were developed with SuperSignal Chemiluminescent Substrate (Thermo, USA) and read using the LAS‐500 equipment (GE, USA). Primary antibodies used were rabbit anti‐DRP1 (ab184247, Abcam, 1:1000), mouse anti‐TOM20 (sc‐17,664, Santa Cruz, 1:1000) and mouse anti‐β‐actin (A5441, Sigma, 1:10000). Secondary antibodies used were anti‐mouse (A9044, Sigma, 1:5000) and anti‐rabbit (SAB3700861, Sigma, 1:5000).