Anti brn2

Protein lysates were prepared by washing cells with 1 ml of cold PBS and resuspended in lysis buffer [150 mM NaCl, 50 mM tris-HCl (pH 8.0), 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, and protease inhibitors] for 30 min at 4°C. Lysates were centrifuged at 5°C for 15 min at 13,000 rpm to remove insoluble debris. Protein concentrations were quantified using Pierce BCA (Thermo Fisher Scientific). Proteins were separated by electrophoresis on an SDS–polyacrylamide gel electrophoresis gel (Bio-Rad), transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific), and blocked with 5% milk in tris-buffered saline (TBS) with Tween 20. The membranes were immunoblotted with anti–c-Myc (Santa Cruz Biotechnology, sc-40), anti–L-Myc (University of Iowa, PCRP-MYCL1-1A3), anti-vinculin (Sigma-Aldrich), anti-ASCL1 (BD Biosciences, #556604), anti-NeuroD1(Proteintech, 12081-1-AP), anti-Smad2 (Cell Signaling Technology, #5339), anti-Rest (Millipore Sigma, 07-579), anti-Hes1 (Santa Cruz Biotechnology, sc-166410), anti-Brn2 (Cell Signaling Technology, 12137), anti-YAP1 (Proteintech, 13584-1-AP). Immunoblots were quantified using ImageJ.