Anti neuritin

Western blotting was conducted largely as previously reported.²⁰, ²⁴ Briefly, SCs were collected and homogenized in ice‐cold lysis buffer. Proteins (60 µg each sample) were separated by 0.1% SDS‐PAGE (Amresco) and electrophoretically transferred to nitrocellulose (Roche). Proteins were incubated in Tris‐buffered saline (5% BSA (Sigma), 5% casein (Sigma), and 0.05% Tween‐20) at 37°C for 1 hour, and then incubated with anti‐neuritin (1:300; R&D systems), anti‐β actin (1:5000; Sigma), anti‐Bcl‐2 (1:1000; CST, Beverly, Massachusetts, USA) or anti‐Bax (1:1000; CST) overnight at 4°C, respectively. Blots were washed and incubated with horseradish peroxidase‐conjugated anti‐goat IgG (1:5000, Santa Cruz, CA) at 37°C for 1 hour. The protein complexes were quantified using densitometric analysis (LeicaQ570C, Solms, Germany). Data were expressed as neuritin, Bcl‐2 or Bax to β‐actin ratios (arbitrary units), respectively.

Antibodies were purchased as follows: anti-NeuN (Millipore, MA, USA), anti-BrdU (Abcam, Cambridge, UK), anti-neuritin (R&D systems, MI, USA), anti- beta-actin (anti-β- actin, Santa Cruz, CA, USA), anti-synaptophysin (Millipore, MA, USA), anti-glial fibrillary acidic protein (anti-GFAP, Dakocytomation, Glostrup, Denmark) antibodies. Neuritin peptide was purchased from Abcam.