Cnpase

Antibodies against P-Stat3 (S727, 1:1000), P-Stat3 (Y705, 1:1000), total Stat3 (1:1000), Bcl-2 (1:1000), caspase-3 (1:1000), CNPase (1:1000) and MBP (1:1000) were from Cell Signaling Technology (Danvers, MA). Antibodies for Cre (1:2000) and β-actin (1:10000) were purchased from Sigma. Western blotting analysis was carried out as described (Kim et al., 2012 (link); Steelman et al., 2013 (link)). Briefly, cells were lysed on ice in lysis buffer (1% Triton X-100, 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Na₂P₂O₇, 10 mM NaF, 1 mM Na₃VO_4, 1 mM PMSF) containing proteinase inhibitor mixture I (Roche Applied Science), sonicated, and then centrifuged to remove cellular debris. Proteins were separated on polyacrylamide gels by SDS-PAGE, transferred to PVDF membranes, blocked with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk or 1% bovine serum albumin (BSA) and probed with the primary antibodies overnight at 4 °C. The membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h at RT and visualized by chemiluminescence using the West Pico ECL reagent (Thermo Scientific, Rockford, IL). Western blot images were acquired with a Bio-Rad Chemidoc XRS gel documentation system and quantified with Quantity One software.

Cic^−/− or Cic^+/+ NSCs were cultured for 7 days in 8 chamber vessel glass slides (BD Falcom, ref# 354108) in either complete proliferation medium (Stemcell Technologies, Cat#05702) supplemented with EGF (20ng/ml) and FGF (10ng/ml) or differentiation medium (Stemcell Technologies, Cat#05704). Cells were fixed by formalin, blocked with blocking buffer (1%BSA,10% goat serum in PBS), and stained with the following primary antibodies: Nestin (Stemcell Technologies, Cat#60051, 1:50), GFAP (Stemcell Technologies, Cat#60048, 1:200 or Cell Signaling Cat#12389, 1:100), Olig2 (Millipore, Cat#AB9610, 1:100), Ki67 (BD, Cat#550609, 1:100), β3 tubulin (Stemcell Technologies, Cat#01409, 1:1000), or CNPase (Cell Signaling Cat#5664, 1:100). After blocking with primary antibodies, cells were washed 3 times in PBS and stained with appropriate Alexa Fluor secondary antibodies (1:500) for 30 minutes at room temperature. Cells were washed with PBS and counterstained with DAPI before being mounted with mounting medium (Vectashield H-1400). Slides were imaged with a Nikon ECLIPSE TE2000-E fluorescent microscope.