Ab281588

The cell suspension was centrifuged at 300 x g for 5 min at room temperature, and the supernatant was discarded, total protein was extracted from cells using lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) with protease inhibitors PMSF(ST506; Beyotime) and kept on ice for 30 min. Protein concentrations were determined by Bradford assay and then 20 µg each protein sample were boiled for 5 min, separated by 10% SDS-PAGE, transferred onto a PVDF membrane, blocked with 5% skim milk in TBST buffer (20% Tween) for 3 h at room temperature and then incubated with primary antibodies at 4˚C overnight. Primary antibodies were Nestin (1:1,000, ab6320; Abcam), Sox2 (1:1,000, ab171380; Abcam), GFAP (1:1,000, ab279290; Abcam), Map2 (1:1,000, ab281588; Abcam), ERRα (1:1,000, bs-6998R; Bioss), TGF-β1 (1:1,000, ab215715; Abcam), α-tubulin (1:10,000, ab7291; Abcam), AURKB (1:10,000, ab45145; Abcam) and Id2 (1:500, ab90055; Abcam). The membranes were then washed three times with TBST and incubated with a secondary antibody (1:30,000, Goat Anti-Mouse IgG H&L/HRP, bs-40296G-HRP; 1:30,000, Goat Anti-Rabbit IgG H&L/HRP antibody, bs-40295G-HRP; Bioss) at room temperature for 1 h. Protein bands were visualized using ECL (MilliporeSigma).