Immunoblotting Technique for Protein Analysis

Immunoblotting was performed as described in our previous reports [31 (link), 41 (link), 56 (link), 58 (link)]. In brief, cells were lysed and total protein was quantified using the Bradford assay (Bio-Rad Laboratories Inc.). An equal amount of total protein (20–25 μg) in each sample was loaded onto an SDS-PAGE gel. After transferring to PVDF membrane, the blot was incubated with antibodies. Antibodies were diluted as follows: anti-Nestin (Santa Cruz Biotechnology, Inc., 1:200), anti-NOTCH1 (Cell Signaling Technology, 1:1000), anti-GFAP (Cell Signaling Technology, 1:1000), anti-PDGFRA (Cell Signaling Technology, 1:1000), anti-B-Actin (SigmaAldrich Co. LLC, 1:10000), and anti-GAPDH (Santa Cruz Biotechnology, Inc., 1:1000). Images were taken using a ChemiDoc MP System (Bio-Rad Laboratories Inc.).