Pe conjugated mouse anti human cd133

The DAB2IP expression plasmid was prepared as described previously [22 (link)], and stable clones were selected by G418 at 1000 µg/mL for 3–4 wk. Knock-down of DAB2IP gene was performed using pGIPZ-DAB2IP-lentiviral-shRNAmir and pGIPZ-non-silencing-lentiviral-shRNAmir purchased from Open Biosystems, according to the manufacturer’s protocol. Following infection, cells were selected by puromycin at 0.2 µg/mL for 3–4 wk. The NLGN3 expression plasmid (CAG-HA-NLGN3 WT) was acquired from Peter Scheiffele (Addgene plasmid #59318; http://n2t.net.addgene:59318; RRID:Addgene_59318) [35 (link)]. Primary antibodies used were as follows: anti-NLGN3 (NBP1–90080, N-terminal epitope) was purchased from NOVUS biologicals; anti-DAB2IP (ab87811), anti-NLGN3 (ab186307, C-terminal epitope), and anti-NRXN3 (ab230635) were from Abcam; and anti-EGFR (Cat#4267), anti-HDAC2 (Cat#57156), anti-HSP90 (Cat#4874), anti-phospho-β-Catenin (Cat#9565), anti-phospho-GSK-3β (Cat#5558), and anti-actin (Cat#4970) were purchased from Cell Signaling Technology. PE-conjugated mouse anti-human CD133 (566593) was purchased from BD Pharmingen.

Tumor cells stained with PE‐conjugated mouse anti‐human CD133, APC‐conjugated mouse anti‐human CD49f (BD Biosciences, San Jose, CA, USA), and unstained cells control were subject to flow cytometric analysis. Data were acquired on a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Inc., Ashland, OR, USA).