Pierce fast western kit

Rabbit anti-OLIG2 antibody was purchased from Millipore, CA (Cat. AB9610). Anti-GAPDH antibody was purchased from Millipore (Cat. ABS16). Pierce Fast Western Kit was purchased from Thermo Fisher Scientific Inc, MA (Cat. 35601). The PCR-cloned full-length Olig2 3′ UTR sequence from NSCs cDNA was sub-cloned downstream of pZsGreen1-C1 (Clontech) plasmid to generate the ZsGreen1 reporter constructs. The pCMV-Sport6 plasmid was purchased from Horizon Discovery and the Olig2 cDNA was purchased from Dharmacon and cloned into the pCMV-Sport6 vector for overexpression assays. The plasmid pcDNA3.1 mRuby was obtained from Dr. Jianbo Wu from Dr. Radbod Darabi lab.

Cells were lysed using RIPA buffer for protein extraction. The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Bio-Rad, USA). An equal amount of protein was separated by 8% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris Buffered Saline-0.1% Tween 20 (TBST)-3% bovine serum albumin (Sigma-Aldrich) and then incubated overnight at 4 °C with primary antibodies against p-AKT (rabbit polyclonal antibody, 1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), AKT (rabbit polyclonal antibody, 1:1000, Cell Signaling Technology, Inc.), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; rabbit polyclonal antibody, 1:2000, Santa Cruz Biotechnology). Subsequently, the membranes were washed with TBST solution and then incubated with HRP-conjugated secondary antibody (1:2000). Protein bands were visualized with Pierce™ Fast Western Kit (Thermo Fisher Scientific) and quantified by ImageJ software (National Institutes of Health).