Anti mbp

CC and cortices from P7, P15 and P30 WT and JNK1KO mice, after brain sectioning with Leica vibratome, were obtained by dissection. Tissue lysates were obtained adding RIPA buffer (1% NP40, 150 mM NaCl, 50 mM TRIS HCl pH 8, 5 mM EDTA, 0.01% SDS, 0.005% Sodium deoxycholate, Roche protease inhibitors, PMSF) for 10 min at 4 °C. Samples were homogenized on ice with a pellet pestle (Sigma-Aldrich, Saint Louis, MS, USA) and centrifuged at 1300 rpm at 4 °C. For immunoblots, equal amounts of proteins were resolved by SDS–PAGE and blotted to nitrocellulose membranes, which were then probed with anti-MBP (1:1000, Millipore, Billerica, MS, USA—MW: 18–21 kDa), -CNPase (1:500, Sigma-Aldrich, Saint Louis, MS, USA—MW: 47 kDa), -MOG (1:1000, Proteintech, Manchester, UK—MW: 25 kDa) and -SMI31 (1:1000, SMI-31R Sternberger—MW: 160–200 kDa) antibodies. The membranes were subsequently incubated with the secondary antibodies and developed using the Luminata Forte HRP substrate (Millipore, Billerica, MS, USA). Signals are normalized using anti-β-Tubulin (1:5000, Sigma-Aldrich, Saint Louis, MS, USA—MW: 50 kDa) and anti-Vinculin (1:2000, Sigma-Aldrich, Saint Louis, MS, USA—MW: ~ 120 kDa) antibodies. Blots were imaged on a ChemiDoc (Bio-Rad) and analyzed using Image Lab software.

Antibodies used in this study are as follows: anti-CNPase (Sigma, St. Louis, MO, USA); anti-APC (Millipore, Billerica, MA, USA); anti-MBP (Millipore); anti-β-tubulin (Sigma); anti-GFP (Abcam, Cambridge, MA, USA); anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); HRP-linked anti-IgG (Cell Signaling, Danvers, MA, USA); Alexa-Flour568-anti-IgG and Alexa-Flour488-anti-IgG (Molecular Probes). DAPI was purchased from Invitrogen.