Anti nestin sc 23927

Differentiated cells (adherent cells) and GSCs of the U87MG cell line were evaluated by flow cytometry (BD C6 Accuri and BD FACSJazz™, USA). Briefly, 1.0 × 10⁶ cells were fixed with PFA 3.7% for 15 min at room temperature and permeabilized with cold methanol for 45 min at 4°C. Cells were then blocked for 45 min with 1 × PBS-BSA 0.5% at room temperature. Lastly, the cells were marked with the anti-MRP1 (sc-18835, Santa Cruz), anti-A₃AR (sc-13938, Santa Cruz), anti-Nestin (sc-23927, Santa Cruz), and anti-CD133 (130-092-395, Miltenyi Biotec) antibodies at 4°C overnight and were detected by flow cytometry using their respective Alexa fluor 488 antibody (Life Technology) through the FL1 filter (530/30 nm) of the cytometer.

AMG232 and RG7112 were purchased from APExBIO Technology. Anti-p21 (#2947), anti-PARP (#9542), and anti-ZEB1 (#3396) were purchased from Cell Signaling, anti-p53 (sc-6243), anti-MDM2 (sc-813), and anti-Nestin (sc-23927) were purchased from Santa Cruz Biotechnology and anti-beta-actin (ab8227) was purchased from Abcam. Alexa Fluor 488 anti-rabbit secondary antibody (A11008) was purchased from Thermo Fisher Scientific. Laminin (L2020) was purchased from Sigma and Hoechest33342 (H3570) was purchased from Life Technologies.

Cells were lysed in buffer containing 1% Nonidet P-40, 1 mmol/liter EDTA, 50 mmol/liter Tris-HCl (pH 7.5), and 150 mmol/liter NaCl supplemented with Complete protease inhibitors (Roche). Total proteins were resolved in a 10-12% polyacrylamide gel under denaturing conditions and transferred to nitrocellulose filters for Western blot analyses. Membranes were blocked with 5% BSA in TBS and incubated with the primary antibodies. Membranes were then incubated with the horseradish peroxidase–conjugated secondary antibody (1:3.000) and the reaction was detected with a Western blotting detection system (enhanced chemiluminescence; GE Healthcare). The primary antibodies used were: anti-PATZ1 [22 (link)]; anti-NESTIN (sc-23927, Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDGFRβ (3169, Cell Signaling Technology, Boston, MA); anti-NANOG (sc-134218, Santa Cruz Biotechnology); anti-SOX2 (sc-20088, Santa Cruz Biotechnology); anti-CXCR4 (C8727, Sigma-Aldrich). To ascertain that equal amounts of protein were loaded, the membranes were incubated with anti-ACTIN (sc-1616-R, Santa Cruz Biotechnology) or anti-VINCULIN (sc-7649, Santa Cruz Biotechnology) antibodies.