Cnpase

Purified OPCs were placed on coverslips coated with poly-l-lysine and laminin (20,000 cells/coverslip), and FGF2 (20 ng/mL; R&D Systems, Minneapolis, MN, USA) or total protein from CT CHO cells or A1-expressing CHO cells (0.1 mg/mL, see above) were added in differentiation medium (BME:F12 media (Gibco | Thermo Fisher Scientific, Waltham, MA USA) [1:1] supplemented with 100 μg/mL transferrin, 20 μg/mL putrescine, 12.8 ng/mL progesterone, 10.4 ng/mL selenium, 25 μg/mL insulin, 0.8 μg/mL thyroxine, 0.6% glucose, and 6.6 mM glutamine, as reported previously [37 (link),41 (link),44 (link)]. AraC (5 µM; Sigma-Aldrich, San Luis, AZ, USA) was added to the cultures to inhibit proliferation and after 5–7 days in vitro (DIV), the cells were fixed for immunocytochemical detection of CNPase (1:200; Covance, Princeton, NJ, USA) and Olig-2 (1:200; Millipore, Burlington, NJ, USA). Finally, 10 random fields per coverslip were photographed under a Leica microscope using a20X objective and the proportion of CNPase⁺ cells was assessed relative to the respective controls (±s.e.m.).

Differentiated cultured NS were re-suspended in ice-cold cell lysis buffer (Cell Signaling Technology) with protease inhibitor cocktail (Roche) and incubated for 15–30 min on ice. A total amount of 30 µg of protein was loaded on a 10% or 12% SDS-PAGE gel and transferred to nitrocellulose membranes (Protran, Whatman). The membranes were blocked in Tris-buffered saline with 0.05% Tween-20 and 5% skimmed milk or 4% BSA (MAP-2 blots), incubated with primary and secondary antibodies, and washed according to standard procedures. Primary antibodies used were: musashi-1 (1/500, rabbit; Abcam), nestin (1/500, rabbit; Abcam), SOX-2 (1/1000, mouse, Cell Signaling), ki67 (1/500, rabbit, Abcam), PCNA (1/500, mouse, Millipore), β-III-tubulin (1/1000, TuJ clone; mouse; Covance), MAP-2 (1/500, mouse; Sigma), CNPase (1/500, rabbit, Covance), GFAP (1/1000, mouse; Sigma) and α-tubulin (1/5000, mouse; Sigma). Secondary peroxidase-conjugated (1/1000) donkey anti-rabbit (Amersham Biosciences, GE Healthcare), or rabbit anti-mouse antibodies (Jackson Immunoresearch) were used. Values in figures are the average of the quantification of at least three independent experiments each of them corresponding to four different cellular pools.