, 2007, Bull and Bartlett, 2005, Luo et al., 2010 and Seaberg and van der Kooy, 2002). NPC maintenance, proliferation and differentiation Anticancer Compound Library analyses, and neurosphere assays were performed as described in our publications ( Barkho et al., 2008, Liu et al., 2010, Luo et al., 2010 and Szulwach et al., 2010). Transfection

of NPCs was performed using a Stemfect kit (Stemgent, San Diego, CA), and luciferase activity was detected using the Dual-Luciferase Reporter 1000 System (Promega, # E1980), based on the manufacturer’s protocols and our publications (Liu et al., 2010 and Smrt et al., 2010). For in vivo acute knockdown of FXR2, retroviral grating was performed as described (Liu et al., 2010, Smrt et al., 2007, Smrt et al., 2010 and Szulwach et al., 2010). RNA-IP was performed as described (Brown et al., 2003 and Luo et al., 2010). WT and Fxr2 KO NPCs (2 × 106) and a monoclonal antibody against FXR2 (F1554, Sigma-Aldrich) Selleck C59 wnt were used. RT-PCR

and real-time PCR were performed using standard methods as described (Liu et al., 2010 and Luo et al., 2010). Differential gene expression in Fxr2 KO DG-NPCs was determined using mouse Neural Stem Cell Pathway Arraya (QIAGEN) according to the manufacturer’s instructions (QIAGEN). DG-NPCs were treated with 10 μg/ml of actinomycin D (Sigma-Aldrich) to inhibit gene transcription, based on a published method (Ghosh et al., 2009), and cells were collected at various time intervals. Noggin mRNA levels normalized to GAPDH were assessed by real-time PCR. Performance of this

procedure was based on a previously published method with minor modifications (Deschenes-Furry et al., 2007). To determine the amount of secreted Noggin protein in the cell medium, medium was collected after 24 hr of culture. Determination of secreted Noggin was performed using a mouse Noggin ELISA kit (ABIN425343, antibodies-online.com, else Atlanta, USA), according to the manufacturer’s instructions. For growth factor and antibody treatment, the concentration of Noggin was 250 ng/ml (R&D Systems), BMP2 was 25 ng/ml (R&D Systems), and Noggin antibody was 2.5 ng/ml (R&D Systems). Conditioned medium was collected from WT and KO DG-NPCs after 24 hr in culture. Lentivirus expressing control shRNA (shCon) was published previously (Barkho et al., 2008 and Liu et al., 2010). Noggin-shRNA plasmids were purchased (QIAGEN), and their efficiency at knocking down endogenous Noggin was tested by transfecting P19 cells followed by Western blotting analyses. The shRNA exhibiting highest efficacy was then cloned into the lentiviral vector, and in vivo lentiviral grafting was performed as described (Liu et al., 2010, Smrt et al., 2007, Smrt et al., 2010 and Szulwach et al., 2010). Based on the published criteria (Clelland et al.